FTO Inhibitors

ABSTRACT

The invention provides compounds that inhibit FTO (fat mass and obesity), including pharmaceutically acceptable salts, hydrides and stereoisomers thereof. The compounds are employed in pharmaceutical compositions, and methods of making and use, including treating a person in need thereof, particularly obesity, with an effective amount of the compound or composition, and detecting a resultant improvement in the person&#39;s health or condition.

INTRODUCTION

Obesity is a severe health problem worldwide and many factors contributeto this chronic disease, including environmental factors and geneticfactors. Genome-wide association studies to investigate patients withobesity revealed a gene for FTO (fat mass and obesity) to stronglyassociate with obesity. FTO's functional role in obesity was confirmedin transgenic animal models, such as FTO knockout mouse,FTO-overexpression mouse and FTO-I367F mutation mouse. FTO protein is anα-ketoglutarate and iron (II) dependent nucleic acid demethylase. Itspreferred substrate is N6-meA in message RNA, which locates near thestop codon and influences gene translation.

We disclosed in US2014/0148383A1 identification of a known FDA approveddrug—entacapone as an FTO inhibitor using structure-based virtualscreening method in combination with biological activity measurements,including enzymatic activity, cellular activity and in high-fat dietinduced obesity (DIO) animal model. Entacapone is a COMT(Catechol-O-methyltransferase) inhibitor used for treating Parkinsondisease.

We synthesized numerous derivative and analogs, however activity assaysrevealed many substitutions reduced or obliterated FTO inhibitoryactivity, discouraging conventional SAR investigation. Undeterred wepursued a radical derivitization program introducing disruptivefunctional groups. Here we disclose a novel structural class of FTOinhibitors, composition and methods of use.

EP1978014 discloses processes for preparing entacapone (I) bydemethylation of dimethoxy-entacapone (II), wherein II may be preparedby reacting a hydroxyl intermediary (III) with MHB(OCOR)₃. This hydroxylintermediary is coincidentally structurally related to some of thesubject compounds.

SUMMARY OF THE INVENTION

The invention provides compounds, compositions and methods forinhibiting FTO and treating disease associated with excess FTO activity,including obesity, obesity-related diseases and Alzheimer's disease. Inone aspect the invention provides an FTO inhibitor selected from acompound formula I, a stereoisomer thereof, a hydride thereof, and apharmaceutically-acceptable salt thereof, or a pharmaceuticalcomposition formulated and suitable for administration to a person andcomprising in unit dosage the inhibitor:

wherein:(a)R1 and R2 are independently H or Me;R3 is OH or NHR, wherein R is H or an optionally substituted, optionallyhetero-, optionally cyclic C1-C18 hydrocarbyl; andR4 is optionally substituted, optionally hetero-, optionally cyclicC1-C18 hydrocarbyl;(b)R1 and R2 are independently H or Me;R3 is H, OH or NHR, wherein R is H or C1-C4 alkyl, esp. Me;

R4 is CONHR5; and

R5 is optionally substituted, optionally hetero-, optionally cyclicC1-C18 hydrocarbyl;(c)R1 and R2 are independently H or Me;R3 is H, OH or NHR, wherein R is H or C1-C4 alkyl, esp. Me;

R4 is COR5; and

R5 is optionally substituted, heterocyclic C3-C18 hydrocarbyl comprisingan n-membered ring wherein n=3-18 (3, 4, 5, 6, 9 or 10) including 1 ton-1 heteroatoms independently selected from N, O, S and P; or(d)R1 and R2 are independently H or Me;R3 is H, OH or NHR, wherein R is H or C1-C4 alkyl, esp. Me; andR4 is optionally substituted, heterocyclic C3-C18 hydrocarbyl comprisingan n-membered ring wherein n=3-18 (3, 4, 5, 6, 9 or 10) including 1 ton-1 heteroatoms independently selected from N, O, S and P;wherein excluded from the inhibitor, unless present in the composition,are compounds identified by CAS ID number: 309, CAS ID: 1364322-41-7;365, CAS ID:1150310-12-5; 371, CAS ID: 1150310-15-8; and361, CAS ID:143542-72-7, such as if R3 is diethylamide and R4 is OH thenone or both R1 and R2 is H.

In embodiments of the inhibitor or composition the heterocyclic C3-C18hydrocarbyl comprises:

a 3 membered ring that is an optionally substituted: aziridine, oxirane,oxaziridine;a 4 membered ring that is an optionally substituted: azetidine, oxetane,oxazetidine;a 5 membered ring that is an optionally substituted: pyrrole,1,2-diazole (pyrazole), 1,3 diazole (imidazole), thiazole, isothiazole,oxazole, isoxazole, furan, dioxole, thiophene;a 6 membered ring that is an optionally substituted: pyridine, diazine,triazine, oxazine, thiazine, dioxine, oxathiine, dithiine;a 9 membered ring that is an optionally substituted: indole,benzothiazole, benzooxazole, benzofuran, benzodioxole, benzothiophene,benzodithiole; ora 10 membered ring that is an optionally substituted: quinoline,quinoxaline, quinazoline, chromene, benzodioxine, thiochromene,benzodithiine.

In embodiments of the inhibitor or composition the optionallysubstituted, optionally hetero-, optionally cyclic C1-C18 hydrocarbyl ineach instance is an optionally substituted C1-C9 alkyl, C2-C9 alkenyl,C2-C9 alkynyl, or C5-C14 aryl hydrocarbon, comprising 1-5 heteroatomsthat are N, S, O or P, including 1-5 nitrogen atoms, or a heteroatomsubstituted with the hydrocarbon.

In embodiments of the inhibitor or composition:

one or both R1 and R2 is H;R3 is OH; and/orR is H or C1-C4 alkyl, esp. Me.

In an aspect the inventors surprising and unexpectedly found that thecompounds disclosed herein wherein R₃ is OH demonstrated a much longerT_(1/2) and a lower Cl_(int) compared with entacapone, and that theintroduction of hydroxyl imparts better inhibitory activity of FTOreceptor over COMT receptor.

In another aspect the FTO inhibitor is a compound of formula I, supra,or a stereoisomer thereof, a hydride thereof, or apharmaceutically-acceptable salt thereof, wherein:

R₁ and R₂ are each independently H or C₁₋₄alkyl;

R₃ is OH or NHR, wherein R is hydrogen, —C₁₋₄alkyl, cycloalkyl,heterocyclyl, aryl or heteroaryl;

R₄ is hydrogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, —SO₂R_(a), —COR_(a), —CO₂R_(a),—CONR_(a)R_(b), NR_(a)R_(b), —NR_(a)COR_(b), —NR_(a)CO₂R_(b), or—NR_(a)SO₂R_(b); wherein said —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl are each independentlyoptionally substituted with at least one substituent R_(c);

wherein

R_(a) and R_(b) are each independently hydrogen, C₁₋₄alkyl,heteroarylC₁₋₄alkyl-, heterocyclylC₁₋₄alkyl-, aryl, heteroaryl, orC₃₋₆cycloalkyl; or

R_(a) and R_(b), together with the atom(s) to which they are attachedform a 3- or 4- or 5- or 6-membered ring optionally comprising anadditional heteroatom selected from the group of O, NH, S and P; and

R_(c) is hydrogen, halogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, —CN, —NO₂, oxo, —OH,C₁₋₆alkyloxy, —SO₂H, C₁₋₆alkylSO₂ —, —COH, C₁₋₆alkylCO—, CO₂H,C₁₋₆alkylCO₂—, CONH₂ or —NH₂,

provided that said compound is not(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-hydroxyacrylamide.

In another embodiment, the FTO inhibitor is a compound of formula I,supra, or a stereoisomer thereof, a hydride thereof, or apharmaceutically-acceptable salt thereof, wherein:

R₁ and R₂ are each independently H or C₁₋₄alkyl;

R₃ is OH;

R₄ is heteroaryl, —COR_(a), or —CONR_(a)R_(b); wherein said heteroarylis optionally substituted with at least one substituent R_(c);

wherein

R_(a) and R_(b) are each independently hydrogen, C₁₋₄alkyl,heteroarylC₁₋₄alkyl-, heterocyclylC₁₋₄alkyl-, aryl, heteroaryl, orC₃₋₆cycloalkyl; or

R_(a) and R_(b), together with the nitrogen atom to which they areattached form a 3- or 4- or 5- or 6-membered monocyclic ring optionallycomprising an additional heteroatom selected from the group of O, NH, Sand P; and

R_(c) is hydrogen, halogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, —CN, —NO₂, oxo, —OH,C₁₋₆alkyloxy, —SO₂H, C₁₋₆alkylSO₂—, —COH, C₁₋₆alkylCO—, CO₂H,C₁₋₆alkylCO₂—, CONH₂ or —NH₂,

provided that said compound is not(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-hydroxyacrylamide.

In particular embodiments, R₁ and R₂ are each H; one of R₁ and R₂ is H,the other is C₁₋₄alkyl, preferably methyl; or R₁ and R₂ are eachC₁₋₄alkyl, preferably methyl.

In particular embodiments, R₄ is —COR_(a), wherein R_(a) is heteroaryl,wherein preferably R_(a) is a 5-membered heteroaryl comprising onenitrogen atom and one sulfur atom, preferably, R_(a) is thiazolyl, e.g.,thiazole-4-yl.

In embodiments R₄ is —CONR_(a)R_(b), R_(a) and R_(b) are eachindependently hydrogen, C₁₋₄alkyl, heteroarylC₁₋₄alkyl-,heterocyclylC₁₋₄alkyl-, heteroaryl, or C₃₋₆cycloalkyl, whereinpreferably, R_(a) and R_(b) are both C₁₋₄alkyl; more preferably, R_(a)and R_(b) are both ethyl. Alternatively, one of R_(a) and R_(b) ishydrogen, the other is C₁₋₄alkyl, heteroarylC₁₋₄alkyl-,heterocyclylC₁₋₄alkyl-, heteroaryl or C₃₋₆cycloalkyl; preferably, one ofR_(a) and R_(b) is hydrogen, the other is C₁₋₄alkyl,pyrimidinylC₁₋₄alkyl- (e.g., pyrimidin-4-ylmethyl), 5- to 10-memberedheteroaryl (e.g., pyridinyl, pyrizinyl, pyrimidinyl, thiazolyl,benzo[d]thiazolyl, thiadiazolyl), or C₃₋₆cycloalkyl (e.g., cyclopropyl,cyclobutyl, cyclopentyl or cyclohexyl). Alternatively, R_(a) and R_(b),together with the nitrogen atom to which they are attached form a 3- or4- or 5- or 6-membered monocyclic ring optionally comprising anadditional heteroatom selected from the group of O, NH, and S; R_(a) andR_(b), together with the nitrogen atom to which they are attached form a3- or 4- or 5- or 6-membered monocyclic ring optionally comprising oneadditional oxygen atom. Preferably, R_(a) and R_(b), together with thenitrogen atom to which they are attached form a 4- or 6-memberedmonocyclic ring optionally comprising one additional oxygen atom. Morepreferably, R_(a) and R_(b), together with the nitrogen atom to whichthey are attached form a 4-membered monocyclic ring, or a 6-memberedmonocyclic ring or a 6-membered monocyclic ring comprising oneadditional oxygen atom. Most preferably, R_(a) and R_(b), together withthe nitrogen atom to which they are attached form a piperidinyl,azetidinyl or 1,3-oxazinanyl.

In embodiments, R₄ is heteroaryl optionally substituted with at leastone substituent R_(c), wherein R_(c) is as defined above; whereinpreferably, R₄ is a 5- or 6-membered monocyclic heteroaryl comprisingone or two or three or four heteroatoms selected from NH, O, S and P(preferably 5- or 6-membered monocyclic heteroaryl comprising one or twoor three or four heteroatoms selected from NH, O and S); or a 9 or10-membered bicylic heteroaryl comprising one or two or three or fourheteroatoms selected from NH, O, S and P (preferably, a 9 or 10-memberedbicylic heteroaryl comprising one or two or three or four heteroatomsselected from NH, O and S). More preferably, R₄ is pyridinyl, pyrizinyloptionally substituted by carboxyl, pyrimidinyl, thiazolyl,benzo[d]thiazolyl, or 1,2,4-thiadiazolyl. Most preferably, R₄ ispyridin-2-yl, pyrizin-2-yl optionally substituted by carboxyl,pyrimidin-4-yl, thiazol-2-yl, benzo[d]thiazol-2-yl, or1,2,4-thiadiazol-5-yl.

In embodiments the inhibitor is of the following Tables. We measuredcompound inhibition activity in a demethylation reaction catalyzed byFTO (US2014/0148383A1). The reaction system was incubated at 37° C. for2 h and stopped by heating at 95° C. for 5 min ssDNA was digested bynuclease P1 and alkaline phosphatase. The concentrations of N6-mA and Awere analyzed by HPLC-MS/MS. When concentration of substrate and enzymeare 0.5 μM and 0.1 μM, respectively, the measured IC50 value ofentacapone against FTO is ˜3 μM.

TABLE 1 Subsection (a) inhibitors, wherein R3 is OH, demonstrating IC50value <10 μM in demethylation reaction catalyzed by FTO; experimentaldetails below.

347

351

352

523

524

525

503

359

374

668

661

658

673

674

722

697

691

692

701

715

711

TABLE 2 Subsection (a) inhibitors, wherein R3 is NHR, demonstrating IC50value <10 μM in demethylation reaction catalyzed by FTO; experimentaldetails below.

347N

351N

352N

523N

524N

525N

503N

359N

374N

668N

661N

658N

673N

674N

722N

697N

691N

692N

701N

715N

711N

711NM

711NE

711NB

TABLE 3 Subsection (b) inhibitors, wherein R4 is CONHR5, demonstratingIC50 value <10 μM in demethylation reaction catalyzed by FTO;experimental details below.

664

684

688

713

709

712

693

801

802

331

803

804

333

805

318

806

366

807

365 CAS ID: 1150310- 12-5

380

374

668

673

674

722

374N

668N

673N

674N

800N

691N

692N

TABLE 4 Subsection (c) inhibitors, wherein R4 is COR5, demonstratingIC50 value <10 μM in demethylation reaction catalyzed by FTO;experimental details below.

808

687

809

317

810

371 CAS ID: 1150310- 15-3

378

660

382

702

811

812

813

814

815

816

817

818

819

820

821

822

823

824

698

675

825

826

827

394

661

658

701

711

715

711N

661N

658N

701N

715N

TABLE 5 Subsection (d) inhibitors, wherein R4 is heterocyclic,demonstrating IC50 value <10 μM in demethylation reaction catalyzed byFTO; experimental details below.

390

656

666

829

315

400

319

389

502

505

395

396

522

655

830

831

518

520

361 CAS ID: 143542- 72-7

517

519

351

352

523

524

525

503

359

697

351N

352N

523N

524N

525N

503N

359N

697N

In another aspect the invention provides a pharmaceutical compositionsuitable for administration to a human and comprising a subject ordisclosed inhibitor.

The compositions may comprise a pharmaceutically-acceptable excipient,be in effective, unit dosage form, and/or comprise another, differenttherapeutic agents for the targeted disease or condition. Inembodiments, the compositions may further comprise or be copackaged orcoformulated with a second, different medicament for inhibiting weightgain, promoting weight loss, reducing serum LDL, cholesterol, LDL-c, ortriglycerides, or treating obesity or an obesity related disease (esp.obesity-related diabetes, hyperglycemia, diabetic nephropathy,hyperlipemia, coronary heart disease, atherosclerosis, hypertension,cardiovascular or cerebrovascular disease) or Alzheimer's disease.

In embodiments:

the medicament is an AD drug that is an acetylcholinesterase inhibitor(esp. tacrine, rivastigmine, galantamine and donepezil) or an NMDAreceptor antagonist (esp. memantine);

the medicament is a medicament for inhibiting weight gain that is a foodintake inhibitor or a food absorption inhibitor;

the medicament is a medicament for inhibiting weight gain that isOrlistat, Sibutramine, Lorcaserin, Rimonabant, Metformin, Exenatide,Pramlintide, phentermine/topiramate, or a pharmaceutically-acceptablesalt thereof;

the medicament is a medicament for reducing serum LDL, cholesterol,LDL-c, or triglycerides, that is atorvastatin (Lipitor), fluvastatin(Lescol), lovastatin (Altoprev, Mevacor), pravastatin (Pravachol),rosuvastatin (Crestor), simvastatin (Zocor), cholestyramine (Prevalite,Questran), colesevelam (Welchol), colestipol (Colestid), ezetimibe(Zetia), ezetimibe-simvastatin (Vytorin), fenofibrate (Lofibra, TriCor),gemfibrozil (Lopid), Niacin (Niaspan), Omega-3 fatty acid (Lovaza), or apharmaceutically-acceptable salt thereof.

the medicament is a diabetes or hypoglycemia medicament, such asglibenclamide, glipizide, gliquidone, gliclazide, glimepiride,glibornuride, repaglinide, nateglinide, metformin, acarbose, voglibose,rosiglitazone, pioglitazone, exenatide, liraglutide, sitagliptin,saxagliptin, vildagliptin, canagliflozin, dapaglifozin, or apharmaceutically-acceptable salt thereof.

In another aspect the invention provides methods of treating a person inneed thereof with an effective amount of the subject inhibitor orpharmaceutical composition, and optionally, detecting a resultantimprovement in the person's health or condition. The methods may alsooptionally include the antecedent step of determining that the person,particularly diagnosing and applicable disease or condition (herein). Inembodiments the invention provides methods and uses of a subjectinhibitor or composition in a person in need thereof, to inhibit FTO,inhibit weight gain, promote weight loss, reduce serum LDL, cholesterol,LDL-c, or triglycerides, or treat obesity or an obesity related diseaseor Alzheimer's Disease.

The invention encompasses all combination of the particular embodimentsrecited herein, as if each had been separately, laboriously recited. Forexample, subsection (a) encompasses combinations wherein: R1 and R2 areH; R3 is NH₂; and R4 is a 6 membered ring that is pyridine, andsubsection (d) encompasses combinations wherein R1 and R2 are Me; R3 isOH; and R4 is 1,3 diazole.

Description of Particular Embodiments of the Invention

The following descriptions of particular embodiments and examples areprovided by way of illustration and not by way of limitation. Thoseskilled in the art will readily recognize a variety of noncriticalparameters that could be changed or modified to yield essentiallysimilar results.

Unless contraindicated or noted otherwise, in these descriptions andthroughout this specification, the terms “a” and “an” mean one or more,the term “or” means and/or and polynucleotide sequences are understoodto encompass opposite strands as well as alternative backbones describedherein. Furthermore, genuses are recited as shorthand for a recitationof all members of the genus; for example, the recitation of (C1-C3)alkyl is shorthand for a recitation of all C1-C3 alkyls: methyl, ethyland propyl, including isomers thereof.

A hydrocarbyl group is a substituted or unsubstituted, straight-chain,branched or cyclic alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl,arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl groupwhich comprises 1-15 carbon atoms and optionally includes one or moreheteroatoms in its carbon skeleton.

The term “heteroatom” as used herein generally means any atom other thancarbon or hydrogen. Preferred heteroatoms include oxygen (O), phosphorus(P), sulfur (S), nitrogen (N), and halogens, and preferred heteroatomfunctional groups are haloformyl, hydroxyl, aldehyde, amine, azo,carboxyl, cyanyl, thocyanyl, carbonyl, halo, hydroperoxyl, imine,aldimine, isocyanide, iscyante, nitrate, nitrile, nitrite, nitro,nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, and sulfhydryl.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which is fully saturated,having the number of carbon atoms designated (i.e. C1-C8 means one toeight carbons). Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.

The term “alkenyl”, by itself or as part of another substituent, means astraight or branched chain, or cyclic hydrocarbon radical, orcombination thereof, which may be mono- or polyunsaturated, having thenumber of carbon atoms designated (i.e. C2-C8 means two to eightcarbons) and one or more double bonds. Examples of alkenyl groupsinclude vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl) and higher homologs and isomersthereof.

The term “alkynyl”, by itself or as part of another substituent, means astraight or branched chain hydrocarbon radical, or combination thereof,which may be mono- or polyunsaturated, having the number of carbon atomsdesignated (i.e. C2-C8 means two to eight carbons) and one or moretriple bonds. Examples of alkynyl groups include ethynyl, 1- and3-propynyl, 3-butynyl and higher homologs and isomers thereof.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from alkyl, as exemplified by—CH₂—CH₂—CH₂—CH₂—. Typically, an alkyl (or alkylene) group will havefrom 1 to 24 carbon atoms, with those groups having 10 or fewer carbonatoms being preferred in the invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, P, Si and S, wherein the nitrogen,sulfur, and phosphorous atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. The heteroatom(s) O,N, P and S may be placed at any interior position of the heteroalkylgroup. The heteroatom Si may be placed at any position of theheteroalkyl group, including the position at which the alkyl group isattached to the remainder of the molecule. Examples include—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH3)—CH₃. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified by —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Accordingly, acycloalkyl group has the number of carbon atoms designated (i.e., C3-C8means three to eight carbons) and may also have one or two double bonds.A heterocycloalkyl group consists of the number of carbon atomsdesignated and from one to three heteroatoms selected from the groupconsisting of O, N, Si and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. Additionally, for heterocycloalkyl, a heteroatom can occupythe position at which the heterocycle is attached to the remainder ofthe molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include 1-(1,2,5,6-tetrahydropyrid-yl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” and “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include alkyl substituted with halogen atoms, which can be thesame or different, in a number ranging from one to (2m′+1), where m′ isthe total number of carbon atoms in the alkyl group. For example, theterm “halo(C1-C4)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Thus,the term “haloalkyl” includes monohaloalkyl (alkyl substituted with onehalogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms ina number ranging from two to (2m+1) halogen atoms, where m′ is the totalnumber of carbon atoms in the alkyl group). The term “perhaloalkyl”means, unless otherwise stated, alkyl substituted with (2m′+1) halogenatoms, where m′ is the total number of carbon atoms in the alkyl group.For example the term “perhalo(C1-C4)alkyl” is meant to includetrifluoromethyl, pentachloroethyl, 1,1,1-trifluoro-2-bromo-2-chloroethyland the like.

The term “acyl” refers to those groups derived from an organic acid byremoval of the hydroxy portion of the acid. Accordingly, acyl is meantto include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl,benzoyl and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. Non-limiting examples of aryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl and 1,2,3,4-tetrahydronaphthalene.

The term heteroaryl,” refers to aryl groups (or rings) that contain fromzero to four heteroatoms selected from N, O, and S, wherein the nitrogenand sulfur atoms are optionally oxidized and the nitrogen heteroatom areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofheteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyland 6-quinolyl.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (as well as thosegroups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR′—SO₂NR′″,—NR″CO₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—SO₂NR′R″, —NR″SO₂R, —CN and —NO₂, in a number ranging from zero tothree, with those groups having zero, one or two substituents beingparticularly preferred. R′, R″ and R″ each independently refer tohydrogen, unsubstituted (C1-C8)alkyl and heteroalkyl, unsubstitutedaryl, aryl substituted with one to three halogens, unsubstituted alkyl,alkoxy or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R′ and R″are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6- or 7-membered ring. For example, —NR′R″is meant to include 1-pyrrolidinyl and 4-morpholinyl. Typically, analkyl or heteroalkyl group will have from zero to three substituents,with those groups having two or fewer substituents being preferred inthe invention. More preferably, an alkyl or heteroalkyl radical will beunsubstituted or monosubstituted. Most preferably, an alkyl orheteroalkyl radical will be unsubstituted. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups such as trihaloalkyl (e.g., —CF₃ and—CH₂CF₃).

Preferred substituents for the alkyl and heteroalkyl radicals areselected from: —OR, ═O, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO₂R′,—NR′—SO₂NR″R′″, —S(O)R′, —SO2R′, —SO₂NR′R″, —NR″SO₂R, —CN and —NO₂,where R′ and R″ are as defined above. Further preferred substituents areselected from: —OR′, ═O, —NR′R″, halogen, —OC(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR″CO₂R′, —NR′—SO₂NR″R′″, —SO₂R′, —SO₂NR′R″,—NR″SO₂R, —CN and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′,—NR′—C(O)NR″R′″, —NR′—SO₂NR″R′″, —NH—C(NH2)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —SO₂R, —SO₂NR′R″, —NR″SO₂R, —N₃, —CH(Ph)₂,perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R, R″ and R′″ are independently selected fromhydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstitutedaryl)oxy-(C1-C4)alkyl. When the aryl group is1,2,3,4-tetrahydronaphthalene, it may be substituted with a substitutedor unsubstituted (C3-C7)spirocycloalkyl group. The(C3-C7)spirocycloalkyl group may be substituted in the same manner asdefined herein for “cycloalkyl”. Typically, an aryl or heteroaryl groupwill have from zero to three substituents, with those groups having twoor fewer substituents being preferred in the invention. In oneembodiment of the invention, an aryl or heteroaryl group will beunsubstituted or monosubstituted. In another embodiment, an aryl orheteroaryl group will be unsubstituted.

Preferred substituents for aryl and heteroaryl groups are selected from:halogen, —OR, —OC(O)R′, —NR′R″, —SR′, —R, —CN, —NO₂, —CO₂R′, —CONR′R″,—C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —S(O)R′, —SO₂R, —SO₂NR′R″, —NR″SO₂R,—N₃, —CH(Ph)₂, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, whereR′ and R″ are as defined above. Further preferred substituents areselected from: halogen, —OR′, —OC(O)R′, —NR′R″, —R′, —CN, —NO₂, —CO₂R′,—CONR′R″, —NR″C(O)R′, —SO₂R, —SO₂NR′R″, —NR″SO₂R, perfluoro(C1-C4)alkoxyand perfluoro(C1-C4)alkyl.

The substituent —CO₂H, as used herein, includes bioisostericreplacements therefor; see, e.g., The Practice of Medicinal Chemistry;Wermuth, C. G., Ed.; Academic Press: New York, 1996; p. 203.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)q-U-, wherein T and U are independently —NH—, —O—, —CH₂— ora single bond, and q is an integer of from 0 to 2. Alternatively, two ofthe substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula -A-(CH2)r-B-,wherein A and B are independently —CH₂—, —O—, —NH—, —S—, —S(O)—,—S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integer of from 1 to3. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CH₂)s-X— (CH₂)t-, where s and t areindependently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—,—S(O)₂—, or —S(O)₂NR′—. The substituent R in —NR′— and —S(O)₂NR′— isselected from hydrogen or unsubstituted (C1-C6)alkyl.

Preferred substituents are disclosed herein and exemplified in thetables, structures, examples, and claims, and may be applied acrossdifferent compounds of the invention, i.e. substituents of any givencompound may be combinatorially used with other compounds.

In particular embodiments applicable substituents are independentlysubstituted or unsubstituted heteroatom, substituted or unsubstituted,optionally heteroatom C1-C6 alkyl, substituted or unsubstituted,optionally heteroatom C2-C6 alkenyl, substituted or unsubstituted,optionally heteroatom C2-C6 alkynyl, or substituted or unsubstituted,optionally heteroatom C6-C14 aryl, wherein each heteroatom isindependently oxygen, phosphorus, sulfur or nitrogen.

In more particular embodiments, applicable substituents areindependently aldehyde, aldimine, alkanoyloxy, alkoxy, alkoxycarbonyl,alkyloxy, alkyl, amine, azo, halogens, carbamoyl, carbonyl, carboxamido,carboxyl, cyanyl, ester, halo, haloformyl, hydroperoxyl, hydroxyl,imine, isocyanide, iscyante, N-tert-butoxycarbonyl, nitrate, nitrile,nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo,sulfhydryl, thiol, thiocyanyl, trifluoromethyl or trifluromethyl ether(OCF3).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein, and suitable for pharmaceutical use. Whencompounds of the invention contain relatively acidic functionalities,base addition salts can be obtained by contacting the neutral form ofsuch compounds with a sufficient amount of the desired base, either neator in a suitable inert solvent. Examples of pharmaceutically acceptablebase addition salts include sodium, potassium, calcium, ammonium,organic amino, or magnesium salt, or a similar salt. When compounds ofthe invention contain relatively basic functionalities, acid additionsalts can be obtained by contacting the neutral form of such compoundswith a sufficient amount of the desired acid, either neat or in asuitable inert solvent. Examples of pharmaceutically acceptable acidaddition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic,succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike. Certain specific compounds of the invention contain both basic andacidic functionalities that allow the compounds to be converted intoeither base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the invention.

In addition to salt forms, the invention provides compounds which are ina prodrug form. Prodrugs of the compounds described herein are thosecompounds that undergo chemical changes under physiological conditionsto provide the compounds of the invention. Additionally, prodrugs can beconverted to the compounds of the invention by chemical or biochemicalmethods in an ex vivo environment. For example, prodrugs can be slowlyconverted to the compounds of the invention when placed in a transdermalpatch reservoir with a suitable enzyme or chemical reagent. Prodrugs areoften useful because, in some situations, they may be easier toadminister than the parent drug. They may, for instance, be morebioavailable by oral administration than the parent drug. The prodrugmay also have improved solubility in pharmacological compositions overthe parent drug. A wide variety of prodrug derivatives are known in theart, such as those that rely on hydrolytic cleavage or oxidativeactivation of the prodrug. An example, without limitation, of a prodrugwould be a compound of the invention which is administered as an ester(the “prodrug”), but then is metabolically hydrolyzed to the carboxylicacid, the active entity. Additional examples include peptidylderivatives of a compound of the invention.

Certain compounds of the invention can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the invention. Certain compounds of theinvention may exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplated bythe invention and are intended to be within the scope of the invention.

Some of the subject compounds possess asymmetric carbon atoms (opticalcenters) or double bonds; the racemates, diastereomers, geometricisomers and specifically designated or depicted chirality is preferredand in many cases critical for optimal activity; however all suchisomers are all intended to be encompassed within the scope of theinvention.

The compounds of the invention may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe invention, whether radioactive or not, are intended to beencompassed within the scope of the invention.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit, to some significant extent, thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician, such as when administered, is sufficient to preventdevelopment of, or alleviate to some extent, one or more of the symptomsof the condition or disorder being treated. The therapeuticallyeffective amount will vary depending on the compound, the disease andits severity and the age, weight, etc., of the mammal to be treated.

The invention also provides pharmaceutical compositions comprising thesubject compounds and a pharmaceutically acceptable excipient,particularly such compositions comprising a unit dosage of the subjectcompounds, particularly such compositions copackaged with instructionsdescribing use of the composition to treat an applicable disease orcondition (herein).

The compositions for administration can take the form of bulk liquidsolutions or suspensions, or bulk powders. More commonly, however, thecompositions are presented in unit dosage forms to facilitate accuratedosing. The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Typical unit dosage forms includeprefilled, premeasured ampules or syringes of the liquid compositions orpills, tablets, capsules, lozenges or the like in the case of solidcompositions. In such compositions, the compound is usually a minorcomponent (from about 0.1 to about 50% by weight or preferably fromabout 1 to about 40% by weight) with the remainder being variousvehicles or carriers and processing aids helpful for forming the desireddosing form.

Suitable excipients or carriers and methods for preparing administrablecompositions are known or apparent to those skilled in the art and aredescribed in more detail in such publications as Remington'sPharmaceutical Science, Mack Publishing Co, NJ (1991). In addition, thecompounds may be advantageously used in conjunction with othertherapeutic agents as described herein or otherwise known in the art,particularly other anti-necrosis agents. Hence the compositions may beadministered separately, jointly, or combined in a single dosage unit.

The amount administered depends on the compound formulation, route ofadministration, etc. and is generally empirically determined in routinetrials, and variations will necessarily occur depending on the target,the host, and the route of administration, etc. Generally, the quantityof active compound in a unit dose of preparation may be varied oradjusted from about 1, 5, 25 or 100 to about 5, 25, 100, 500, 1000 or2000 mg, according to the particular application. In a particularembodiment, unit dosage forms are packaged in a multipack adapted forsequential use, such as blisterpack, comprising sheets of at least 6, 9or 12 unit dosage forms. The actual dosage employed may be varieddepending upon the requirements of the patient and the severity of thecondition being treated. Determination of the proper dosage for aparticular situation is within the skill of the art. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall amounts until the optimum effect under the circumstances isreached. For convenience, the total daily dosage may be divided andadministered in portions during the day if desired.

The compounds can be administered by a variety of methods including, butnot limited to, parenteral, topical, oral, or local administration, suchas by aerosol or transdermally, for prophylactic and/or therapeutictreatment. Also, in accordance with the knowledge of the skilledclinician, the therapeutic protocols (e.g., dosage amounts and times ofadministration) can be varied in view of the observed effects of theadministered therapeutic agents on the patient, and in view of theobserved responses of the disease to the administered therapeuticagents.

The therapeutics of the invention can be administered in atherapeutically effective dosage and amount, in the process of atherapeutically effective protocol for treatment of the patient. Formore potent compounds, microgram (ug) amounts per kilogram of patientmay be sufficient, for example, in the range of about 1, 10, 100, 1000,10000, 20000 ug/kg to about 10, 100, 1000, 10000, 20000 or 80000 ug/kgof patient weight though optimal dosages are compound specific, andgenerally empirically determined for each compound.

In general, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect, for each therapeutic,each administrative protocol, and administration to specific patientswill also be adjusted to within effective and safe ranges depending onthe patient condition and responsiveness to initial administrations.However, the ultimate administration protocol will be regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition and size of the patient as well as compoundspotency, severity of the disease being treated. For example, a dosageregimen of the compounds can be oral administration of from 10 mg to2000 mg/day, preferably 10 to 1000 mg/day, more preferably 50 to 600mg/day, in two to four (preferably two) divided doses. Intermittenttherapy (e.g., one week out of three weeks or three out of four weeks)may also be used.

In particular embodiments thereof, the person to be treated has agenotype associated with obesity or pathogenic or medically-undesirableweight gain, such as SNP rs7202116 (G), rs1421085 (C), or rs9939609 (A),or a surrogate or proxy SNP in linkage disequilibrium therewith (withrespect to the correlative phenotype; see references below) and having ar² value greater than 0.5; and/or (f) pathogenically expresses orover-expresses FTO or Fto (e.g. comprises and expresses a multi-copy ftogene). Re rs7202116 G, see e.g. Yang et al. , FTO genotype is associatedwith phenotypic variability of body mass index, Nature, Sep. 16, 2012,doi: 10.1038/nature11401 [epub]; re rs9939609 A, see e.g. Freathy R M,et al (2008). “Common variation in the FTO gene alters diabetes-relatedmetabolic traits to the extent expected, given its effect on BMI”.Diabetes 57 (5): 1419-26. doi:10.2337/db07-1466. PMC 3073395. PMID18346983; re rs1421085 C, see e.g. Dina C, et al., (2007). “Variation inFTO contributes to childhood obesity and severe adult obesity”. NatureGenetics 39 (6): 724-6. doi:10.1038/ng2048. PMID 17496; and formulti-copy fto gene mouse, see e.g. Church et al., Overexpression of Ftoleads to increased food intake and results in obesity, Nature Genetics,published online 14 Nov 2010, doi:10.1038/ng.713.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein, including citations therein, are herebyincorporated by reference in their entirety for all purposes.

EXAMPLES: COMPOUND PREPARATION

Compound 347:

347 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide (2)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (1.0 g, 4.4 mmol) in toluene (11 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (10 mL).

Under a nitrogen atmosphere, 60% NaH (0.35 g, 8.8 mmol) was added tosolution of 2-cyano-N,N-diethylacetamide (0.56 g, 4.0 mmol) in anhydrousTHF (15 mL) at −5° C. The resulting suspension was stirred at −5° C. for15 min and the THF solution of 3,4-dimethoxy-5-nitrobenzoyl chloride wasadded over 10 min and stirred for an additional 1 h at −5° C. Thereaction mixture was warmed to 0° C., quenched by the addition of 1N.HClsolution (4 mL) and stirred for 10 min at room temperature, extracted byethyl acetate (25 mL×2), the organic layers was dried with Na₂SO₄ andconcentrated in vacuo to give the title compound as an orange solid (705mg, 99%). MS [MH]⁺ calcd for C₁₆H₁₉N₃O₆ 350.1, found 350.1.

Step 2: Synthesis of2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide (3)

A solution of2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide(500 mg, 1.43 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ inDCM (5 mL, 5 mmol) at −15° C. under a nitrogen atmosphere. The resultingred suspension was stirred for 1 h at −15° C. and allowed to warm toroom temperature overnight. The reaction was quenched by the addition ofH₂O (2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL×3). The organic layers were combined, washed withbrine and dried over Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLC(0.5% TFA, MeOH/H₂O) gave the desired product as a bright yellow solid(80 mg, 17%). ¹H NMR (400 MHz, CDCl₃) δ 10.92 (s, 1H), 8.28 (d, J=2.0Hz, 1H), 7.74 (d, J=1.9 Hz, 1H), 7.26 (s, 3H), 5.93 (s, 1H), 3.66 (d,J=6.0 Hz, 3H), 1.33 (t, J=7.0 Hz, 6H). MS [MH]⁺ calcd for C₁₄H₁₅N₃O₆322.0, found 322.0.

Compound 315:

315 was prepared in one synthetic step from3,4-dihydroxy-5-nitrobenzaldehyde, according to the following procedure:

Step 1: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(pyridin-2-yl)acrylonitrile (2)

A solution of 2-(pyridin-2-yl)acetonitrile (142 mg, 1.2 mmol),3,4-dihydroxy-5-nitrobenzaldehyde (182 mg, 1 mmol) and NH₄OAc (462 mg, 6mmol) in MeOH (10 mL) was heated to reflux for overnight. LCMS showed no3,4-dihydroxy-5-nitrobenzaldehyde left. The reaction mixture was cooledto room temperature. The solid was filtered and washed by MeOH and H₂O.The solid was re-dissolved in MeOH (5 mL). 5 mL of 1N aqueous HCl wasadded to adjust pH 3-4. The desired product was obtained by filter as abright solid (56 mg, 20%). ¹H NMR (400 MHz, DMSO) δ 8.57 (m, 1H), 8.12(s, 1H), 7.92 (d, J=2.2 Hz, 1H), 7.84 (td, J=7.8, 1.8 Hz, 1H), 7.70 (d,J=8.1 Hz, 1H), 7.56 (d, J=2.4 Hz, 1H), 7.31-7.25 (m, 1H). MS [MH]⁺ calcdfor C₁₄H₉N₃O₄ 284.0, found 284.0.

Compound 361:

361 was prepared in four synthetic steps from malonamide, according tothe following procedure:

Step 1: Synthesis of 2-(4,6-dihydroxypyrimidin-2-yl)acetamide (2)

To a solution of NaOEt (21% in EtOH, 167 mL, 450 mmol) in EtOH (170 mL)was added malonamide (22.9 g, 224 mmol). After being refluxed for 2hours, half of EtOH was removed under reduced pressure and theprecipitated solid was filtered and dried under high vacuum forovernight. The dried solid sodium salt (24 g) was dissolved in ice-coldH₂O (70 mL) and brought to pH 2-3 using 3N. HCl (50 mL),recrystallization from water gave2-(4,6-dihydroxypyrimidin-2-yl)acetamide as a pale yellow solid (6.28 g,33%).

Step 2: Synthesis of 2-(4,6-dichloropyrimidin-2-yl)acetonitrile (3)

To a solution of 2-(4,6-dihydroxypyrimidin-2-yl)acetamide (6.28 g, 37.1mmol) in POCl₃ (19 mL, 204 mmol) was placed in a flask which was thenattached to a reflux condenser. Through the condenser was addedN,N-dimethylaniline (10 mL, 79 mmol). The mixture was warmed cautiouslyin an oil bath which is quickly removed when the reaction began. Afterthe initial vigorous reaction had subsided, the reaction was refluxedfor ten minutes longer. The hot material was poured over 100 g ice andthe resulting suspension was extracted (DCM). The combined organiclayers were dried (Na₂SO₄) and concentrated under reduced pressure. Theproduct was purified by column chromatography (SiO₂, PE/EA=4/1) toprovide the desired product as a yellow solid (5.1 g, 27.1 mmol). MS[MH]⁺ calcd for C₆H₃Cl₂N₃ 189.0, found 189.0.

Step 3: Synthesis of 2-(pyrimidin-2-yl)acetonitrile (4)

To a solution of 2-(4,6-dichloropyrimidin-2-yl)acetonitrile (2.2 g, 11.7mmol) and triethylamine (3.0 mL, 20.8 mmol) in ethyl acetate/MeOH (1/1,40 mL) was added 10% Pd/C (400 mg) and the solution was vigorouslystirred for 2.5 hours under H₂ atmosphere (1 atm). The reaction wasfiltered through celite and washed the celite with MeOH. The combinedfiltrates were concentrated under reduced pressure and purified by flashchromatography (SiO₂, PE/EA=1/1) to give the2-(pyrimidin-2-yl)acetonitrile as a pale red liquid (618 mg, 54%). MS[MH]⁺ calcd for C₆H₅N₃ 120.1, found 120.1.

Step 4: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(pyrimidin-2-yl)acrylonitrile (6)

A solution of 2-(pyrimidin-2-yl)acetonitrile (120 mg, 1 mmol),3,4-dihydroxy-5-nitrobenzaldehyde (182 mg, 1 mmol) and NH₄OAc (462 mg, 6mmol) in MeOH (10 mL) was heated to reflux for 5 hours. LCMS showed nostarting materials left. The solid was filtered and washed by MeOH andH₂O, then dissolved in MeOH (5 mL). 5 mL of 1N.HCl was added to adjustpH 3˜4, the solid was filtered and dried in vacuo to give the desiredproduct as a bright yellow solid (250 mg, 88%). ¹H NMR (400 MHz, DMSO) δ8.78 (d, J=4.8 Hz, 2H), 8.35 (s, 1H), 7.96 (d, J=2.5 Hz, 1H), 7.59 (d,J=2.5 Hz, 1H), 7.32 (t, J=4.8 Hz, 1H). MS [MH]⁺ calcd for C₁₃H₈N₄O₄285.0, found 285.0.

Compound 395:

395 was prepared in four synthetic steps from 4-methylpyrimidine,according to the following procedure:

Step 1: Synthesis of 4-(chloromethyl)pyrimidine (2)

4-methylpyrimidine (53.1 mmol, 5 g) was dissolved in CHCl₃ (100 mL), themixture was heated to 75° C., then1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione (26.6 mmol, 6.2 g) wasadded slowly in two portions. The mixture was stirred at 75° C.overnight. After the completion of the reaction, it was filtered andconcentrated in vacco. The residue was purified by column chromatograph(silica gel, PE/EA=30/1 to 10/1) to obtain the desired product (1.64 g,24%) as a yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ(ppm) 9.16 (s, 1H), 8.77(d, J=5.2 Hz, 1H), 7.54 (d, J=5.1 Hz, 1H), 4.60 (s, 2H); MS [MH]+ calcdfor C₅H₆ClN₂ 129.0, found 129.1;

Step 2: Synthesis of 2-(pyrimidin-4-yl)acetonitrile (3)

Anhydrous potassium carbonate (7.78 mmol, 1.08 g), sodium iodide (3.89mmol, 583 mg) and trimethylsilanecarbonitrile (5.83 mmol, 579 mg) weredissolved in acetonitrile (12 mL), the mixture was heated to 50° C.Finally 4-(chloromethyl)-pyrimidine (3.89 mmol, 500 mg) was dropped intothe reaction mixture. The mixture was stirred 50° C. for 2 hours. Thenit was concentrated in vacco and the residue was purified by columnchromatograph (silica gel, PE/EA=1/1) to obtain the desired product (120mg, 26%) as a black oil. ¹H-NMR (400 MHz, CDCl₃) δ (ppm) 9.21 (s, 1H),8.81 (d, J=5.2 Hz, 1H), 7.52 (d, J=5.0 Hz, 1H), 3.94 (s, 2H); MS [MH]+calcd for C₆H₆N₃ 120.1, found 120.2.

Step 3: Synthesis of3-(3,4-dihydroxy-5-nitrophenyl)-2-(pyrimidin-4-yl)acrylonitrile (4)

A mixture of 2-(pyrimidin-4-yl)acetonitrile (0.95 mmol, 113 mg),3,4-dihydroxy-5-nitrobenzaldehyde (0.79 mmol, 145 mg) and ammoniumacetate (4.75 mmol, 366 mg) in methanol (8 mL) was stirred at 80° C. for4 hours, then it was filtered and washed with methanol and water toobtain the desired product (200 mg, 89%). ¹H-NMR (400 MHz, DMSO) δ(ppm)9.08 (s, 1H), 8.72 (d, J=5.5 Hz, 1H), 8.31 (s, 1H), 8.02 (s, 1H), 7.74(d, J=5.5 Hz, 1H), 7.59 (s, 1H), 7.08 (s, 2H); MS [MH]⁻ calcd forC₁₃H₇N₄O₄ 283.1, found 283.0;

Compound 505:

505 was prepared in three synthetic steps from 3-chloropyridazine,according to the following procedure:

Step 1: Synthesis of tert-butyl 2-cyano-2-(pyridazin-3-yl)acetate (2)

To a solution of 3-chloropyridazine (0.5 g, 4.38 mmol) in NMP (2.5 mL)was added potassium carbonate (1.8 g, 13.15 mmol). Then Cert-Butyl2-cyanoacetate (0.88 mL, 6.14 mmol) was added. The yellow suspension waswarmed up to 80° C. and stirred 3 hours at 80° C. The brown suspensionwas cooled down to room temperature. Then it was added to water (10 mL).The brown solution was acidified with HCl (gas evolution, strongfoaming) There was a precipitation. The suspension was filtrated and thefilter cake was washed with water. The filter cake was dissolved inethyl acetate, dried with Na₂SO₄, filtrated and the organic phaseevaporated to yield 600 mg of desired product as a yellow oil. ¹H-NMR(400 MHz, CDCl₃) δ (ppm): 14.3 (bs, 1 H), 7.68 (dd, 1 H), 7.35 (d, 1 H),1.55 (s, 9H). MS [MH]⁺ calcd for C₁₀H₁₁N₃O₂ 206.1, found 206.1;

Step 2: Synthesis of 2-(pyridazin-3-yl)acetonitrile (3)

The product prepared above was combined with TsOH (142 mg) in toluene(50 mL). After being stirred at refluxing for 12 hours, the reaction wascooled to 25° C., diluted with sat. NaHCO₃ and extracted (10 percentMeOH/CH₂Cl₂×3). The organic layers were washed with brine, dried withNa2SO₄, filtered and concentrated under reduced pressure. Purificationof the crude material by flash chromatography (silica gel, 40-45 percentEtOAc/Hexanes) gave the desired product (87 mg, 17% for two steps) aslight yellow oil. MS [MH]⁺ calcd for C₆H₅N₃ 120.0, found 120.0.

Step 3: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(pyridazin-3-yl)acrylonitrile (4)

A mixture of 2-(pyrazin-2-yl)acetonitrile (87 mg, 0.73 mmol),3,4-dihydroxy-5-nitrobenzaldehyde (0.79 mmol, 145 mg) and ammoniumacetate (366 mg, 4.75 mmol) in methanol (8 mL) was stirred at 80° C. for4 hours. Then it was filtered and washed with methanol and water, anddried in vacuo to obtain the desired product (155 mg, 75%) as a yellowsolid. ¹H NMR (400 MHz, DMSO) δ 10.92 (s, 2H), 9.25 (d, J=4.8 Hz, 1H),8.43 (s, 1H), 8.20 (d, J=8.7 Hz, 1H), 8.08 (d, J=2.0 Hz, 1H), 7.95 (d,J=2.0 Hz, 1H), 7.85 (dd, J=8.7, 4.9 Hz, 1H). MS [MH]⁻ calcd for C₆H₅N₃283.1, found 283.0.

Compound 331:

331 was prepared in one synthetic step from3,4-dihydroxy-5-nitrobenzaldehyde, according to the following procedure:

Step 1: Synthesis of(E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-(thiazol-2-yl)acrylamide(2)

A solution of 2-cyano-N-(thiazol-2-yl)acetamide (184 mg, 1.1 mmol),3,4-dihydroxy-5-nitrobenzaldehyde (200 mg, 1.1 mmol) and NH₄OAc (462 mg,6 mmol) in MeOH (10 mL) was heated to reflux for overnight. LCMS showedno 3,4-dihydroxy-5-nitrobenzaldehyde left. The reaction mixture wascooled to room temperature. The solid was filtered and washed by MeOHand H₂O. The solid was re-dissolved in MeOH (5 mL). 5 mL of 1N aqueousHCl was added to adjust pH 3-4. The desired product was obtained byfilter as a bright solid (90 mg, 25%). ¹H NMR (400 MHz, DMSO) δ 8.17 (s,1H), 7.95 (s, 1H), 7.50 (s, 2H), 7.20 (s, 1H). MS [MH]⁺ calcd forC₁₃H₈N₄O₅S 333.0, found 333.0.

Compound 394:

394 was prepared in four synthetic steps from benzene-1,2-diamine,according to the following procedure:

Step 1: Synthesis of 1-(1H-benzo [d]imidazol-2-yl)ethanone (2)

A mixture of 2-oxosuccinic acid (4.9 g, 37 mmol), benzene-1,2-diamine (4g, 37 mmol) and 4N hydrochloride solution (9 mL) in MeOH (30 mL) wasrefluxed for 7 hours. After the completion of the reaction, it wasconcentrated in vacuo to remove the solvent. The residue was dissolvedin ethyl acetate, washed with aq. sodium bicarbonate and brine. Theorganic layers were combined and concentrated under reduced pressure.The residue was purified by column chromatograph (silica gel, PE/EA=5/1)to obtain the desired product (4 g, 67%). MS [MH]⁺ calcd for C₉H₉N₂O161.06, found 161.1.

Step 2: Synthesis of 1-(1H-benzo [d]imidazol-2-yl)-2-bromoethanone (3)

1-(1H-benzo[d]imidazol-2-yl)ethanone (4 g, 25 mmol) was dissolved intetrachloromethane (50 mL). 1-Bromopyrrolidine-2,5-dione (5.3 g, 30mmol) and 2,2′-(diazene-1,2-diyl)bis(2-methylpropane-nitrile) (411 mg,2.5 mmol) were added. The mixture was stirred at 100° C. for 2 hours,then it was concentrated in vacuo and re-dissolved in ethyl acetate. Theorganic layer was washed with water and concentrated to obtain the crudeproduct (2 g, 33%), which was used in the next step without furtherpurification. MS [MH]⁺ calcd for C₉H₈BrN₂O 238.97, found 239.0.

Step 3: Synthesis of 3-(1H-benzo [d]imidazol-2-yl)-3-oxopropanenitrile(4)

A mixture of 1-(1H-benzo[d]imidazol-2-yl)-2-bromoethanone (2 g, 8.4mmol), trimethylsilane-carbonitrile (1.66 g, 16.7 mmol), TBAF (2.2 g,8.4 mmol) in dichloromethane (15 mL) was stirred at room temperature for24 hours. Then it was concentrated in vacuo and re-dissolved in ethylacetate. The organic layer was washed with water and concentrated toobtain the crude product (400 mg, 26%), which was used in the next stepwithout further purification. MS [MH]⁺ calcd for C₁₀H₇N₃O 186.06, found186.1.

Step 4: Synthesis of 2-(1H-benzo[d]imidazole-2-carbonyl)-3-(3,4-dihydroxy-5-nitrophenyl)acrylo-nitrile(5)

A solution of 3,4-dihydroxy-5-nitrobenzaldehyde (107 mg, 0.59 mmol) and3-(1H-benzo[d]imida-zole-2-yl)-3-oxopropanenitrile (130 mg, 0.7 mmol)and NH₄OAc (273 mg, 3.54 mmol) in methanol (10 mL) was heated to refluxfor overnight. LCMS showed the desired product was formed, the reactionmixture was cooled to room temperature and concentrated in vacuo toremove the solvent. The desired product was obtained by Prep-HPLC (50mg, 24%). ¹H-NMR (400 MHz, DMSO-d⁶) δ (ppm) 12.63 (s, 1H), 8.95 (s, 1H),7.85 (s, 1H), 7.76 (d, J=8.2 Hz, 1H), 7.65 (s, 1H), 7.50 (t, J=7.5 Hz,1H), 7.38-7.26 (m, 2H), 7.12 (s, 2H). MS [MH]⁺ calcd for C₁₇H₁₁N₄O₅351.07, found 351.0.

Compound 382:

382 was prepared in two synthetic steps from ethyl 2-cyanoacetate,according to the following procedure:

Step 1: Synthesis of 3-morpholino-3-oxopropanenitrile (2)

A mixture of sodium ethoxide (0.1 mmol) in ethanol (3 mL), ethylcyanoacetate (1.13 g, 10 mmol) and morpholine (0.85 g, 10 mmol) wasstirred at room temperature for 24 hours. The precipitate was collectedby filtration, washed with diethylether and recrystallised in ethanol toprovide a white solid of 3-morpholino-3-oxopropanenitrile (0.56 g, 35%).

Step 2: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(piperidine-1-carbonyl)acrylonitrile(3)

A solution of 3-morpholino-3-oxopropanenitrile (300 mg, 2.0 mmol),3,4-dihydroxy-5-nitro-benzaldehyde (188 mg, 1.1 mmol) and NH₄OAc (462mg, 6 mmol) in MeOH (10 mL) was heated to reflux for 5 hours. LCMSshowed no 3,4-dihydroxy-5-nitrobenzaldehyde left. The reaction mixturewas cooled to room temperature, concentrated in vacuo to dryness.Further purification by Prep-HPLC (0.5% TFA, MeOH/H₂O) afforded thedesired product as a yellow solid (60 mg, 19%). ¹H NMR (400 MHz, DMSO) δ10.87 (s, 2H), 7.94 (d, J=2.1 Hz, 1H), 7.77 (d, J=2.1 Hz, 1H), 7.68 (s,1H), 3.56-3.66 (m, 8H). MS [MH]⁺ calcd for C₁₄H₁₃N₃O₆ 320.1, found320.0.

Compound 351:

351 was prepared in three synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of 3,4-dimethoxy-5-nitrobenzoyl chloride (2)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. for 15 hours. Thesolvent was removed under reduced pressure. The resulting yellowishsolid (500 mg, 92%) was used in the next step without further workup.

Step 2: Synthesis of3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-2-(pyridin-2-yl)propanenitrile (3)

Under a nitrogen atmosphere, NaH (60% w/w, 176 mg, 4.4 mmol) was addedto solution of 2-(pyridin-2-yl)acetonitrile (236 mg, 2.0 mmol) inanhydrous THF (10 mL) at −5° C. The resulting suspension was stirred at−5° C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoylchloride (500 mg, 2.2 mmol) in THF (5 mL) was added over 10 min andstirred for an additional 1 hour at −5° C. The reaction mixture waswarmed to 0° C., quenched by the addition of 1N.HCl solution (4 mL) andstirred for 10 min at room temperature. The mixture was extracted withethyl acetate (25 mL×2). The combined organic layers were dried withanhydrous sodium sulfate and concentrated in vacuo to give the desiredproduct (425 mg, 64%). MS [MH]⁺ calcd for C16H14N3O5 328.09, found328.1.

Step 3: Synthesis of3-(3,4-dihydroxy-5-nitrophenyl)-3-oxo-2-(pyridin-2-yl)propanenitrile (4)

A solution of3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-2-(pyridin-2-yl)propanenitrile(425 mg, 1.3 mmol) in dichloromethane (5 mL) was added 1.0 M solution ofBBr₃ in dichloromethane (10 mL, 10 mmol) at −15° C. under a nitrogenatmosphere. The resulting suspension was stirred for 1 hours at −15° C.and allowed to warm to room temperature for overnight. The reaction wasquenched slowly by the addition of water (4 mL) and stirred for another30 min. The aqueous phase was extracted with ethyl acetate (30 mL×3).The organic layers were combined, washed with brine and dried over withanhydrous sodium sulfate. The solvent was removed under reduced pressureto give the crude product. Further purification was conducted byPrep-HPLC to obtain the desired product (65 mg, 17%). ¹H-NMR (400 MHz,DMSO-d⁶) δ(ppm) 16.11 (s, 1H), 10.65 (s, 2H), 8.38 (t, J=5.7 Hz, 1H),8.28-7.96 (m, 1H), 7.84 (d, J=2.0 Hz, 1H), 7.55 (d, J=2.1 Hz, 1H), 7.47(d, J=8.7 Hz, 1H), 7.26 (t, J=6.6 Hz, 1H). MS [MH]⁺ calcd for C₁₄H₁₀N₃O₅300.05, found 300.0.

Compound 371:

371 was prepared in two synthetic steps from 2-cyanoacetyl chloride,according to the following procedure:

Step 1: Synthesis of 3-oxo-3-(piperidin-1 -yl)propanenitrile (2)

A mixture of piperidine (5 mL, 50.6 mmol), in DCM (25 mL) was added2-cyanoacetyl chloride (5 mL) at 0° C., then warmed to room temperatureovernight. The reaction mixture was quenched by H₂O, and concentrated invacuo to dryness, the residue was purified by column chromatography(SiO₂, PE/EA=1/1) to give the 3-oxo-3-(piperidin-1-yl)propanenitrile asa yellow oil (500 mg, 7%). MS [MH]⁺ calcd for C₈H₁₂N₂O 153.1, found153.1.

Step 2: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(piperidine-1-carbonyl)acrylonitrile(3)

A solution of 3-oxo-3-(piperidin-1 -yl)propanenitrile (300 mg, 2 mmol),3,4-dihydroxy-5-nitrobenzaldehyde (273 mg, 1.5 mmol) and NH₄OAc (924 mg,12 mmol) in MeOH (15 mL) was heated to reflux for 3 hours. The solid wasfiltered and washed by MeOH and H₂O to give the crude product. The soliddissolved in MeOH (5 mL) was added 1N.HCl (0.5 mL), the color waschanged and the solid was formed, the solid was filtered and washed byH₂O, dried in vacuo to give the(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(piperidine-1-carbonyl)acrylonitrileas a bright yellow solid (60 mg, 13%). ¹H NMR (400 MHz, DMSO) δ 10.86(s, 2H), 7.92 (d, J=2.1 Hz, 1H), 7.75 (d, J=2.0 Hz, 1H), 7.63 (s, 1H),3.56-3.45 (m, 4H), 1.55-1.62 (m, 6H). MS [MH]⁺ calcd for C₁₅H₁₅N₃O₅318.3, found 318.0.

Compound 518:

518 was prepared in two synthetic steps from2-(pyridin-3-yl)acetonitrile, according to the following procedure:

Step 1: Synthesis of 3-(cyanomethyl)pyridine 1-oxide (2)

A solution of 2-(pyridin-3-yl)acetonitrile (625 mg, 5.3 mmol) and m-CPBA(1.36 g, 7.95 mmol) in CHCl₃ (20 mL) was stirred at room temperature forovernight. The reaction mixture was quenched by sat.NaHCO₃ and extractedby DCM and MeOH (DCM/MeOH=10/1). The combined organic layers were driedover anhydrous Na₂SO₄, filtered and dried in vacuo to give the crudeproduct as a white solid (780 mg, >100%), which was used to the nextstep without further purification. MS [MH]⁺ calcd for C₇H₆N₂O 135.0found 135.0.

Step 2: Synthesis of(E)-3-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)pyridine 1-oxide (3)

A solution of 3-(cyanomethyl)pyridine 1-oxide (400 mg, 3.0 mmol),3,4-dihydroxy-5-nitro-benzaldehyde (270 mg, 1.5 mmol) and NH₄OAc (693mg, 9 mmol) in MeOH (10 mL) was heated to reflux for overnight. LCMSshowed no 3,4-dihydroxy-5-nitrobenzaldehyde left. The reaction mixturewas cooled to room temperature. The solid was filtered and washed byMeOH and H₂O. The solid was re-dissolved in MeOH (5 mL). 5 mL of 1Naqueous HCl was added to adjust pH 3-4. The desired product was obtainedby filter as a bright solid (110 mg, 18%). ¹H NMR (301 MHz, DMSO) δ13.71 (s, 1H), 10.89 (s, 2H), 8.65 (s, 1H), 8.26 (d, J=6.2 Hz, 1H), 8.14(s, 1H), 7.99 (d, J=2.0 Hz, 1H), 7.84 (d, J=2.0 Hz, 1H), 7.67-7.50 (m,2H). MS [MH]⁺ calcd for C₁₄H₉N₃O₅ 300.0 found 300.0.

Compound 523:

523 was prepared in three synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of 3,4-dimethoxy-5-nitrobenzoyl chloride (2)

Under a nitrogen atmosphere, SOCl₂ (0.76 mL, 10.56 mmol) and anhydrousDMF (0.02 mL, 0.44 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (1 g, 4.4 mmol) in toluene (20 mL) atroom temperature. The mixture was heated at 60° C. and stirred for 15hours. The solvent was removed under reduced pressure. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride (1 g, 93%) wasused in the next step without further workup.

Step 2: Synthesis of3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(pyrazin-2-yl)acrylonitrile(3)

Under a nitrogen atmosphere, 60% NaH (336 mg, 8.4 mmol) was added tosolution of 2-(pyrazin-2-yl)acetonitrile (500 mg, 4.2 mmol) in anhydrousTHF (5 mL) at −5° C. The resulting suspension was stirred at −5° C. for15 min and the THF solution of 3,4-dimethoxy-5-nitrobenzoyl chloride(500 mg, 4.07 mmol) was added over 10 min and stirred for an additional1 hour at −5° C. The reaction mixture was warmed to 0° C., quenched bythe addition of 1N.HCl solution (8 mL) and stirred for 10 min at roomtemperature, then extracted with ethyl acetate (30 mL×3), the organiclayer was dried with anhydrous sodium sulfate and concentrated in vacuoto obtain the desired product (440 mg, 33%). MS [MH]⁻ calcd forC₁₅H₁₁N₄O₅ 327.08, found 327.1.

Step 3: Synthesis of3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(pyrazin-2-yl)acrylonitrile(4)

To a solution of3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(pyrazin-2-yl)acrylonitrile(200 mg, 0.61 mmol) in anhydrous dichloromethane (5 mL) was added 1.0 Msolution of BBr₃ in dichloromethane (3 mL, 3 mmol) at −15° C. under anitrogen atmosphere. The resulting suspension was stirred for 1 hour at−15° C. and allowed to warm to room temperature for overnight. Thereaction was quenched by the addition of water (2 mL) and stirred foranother 30 min. The aqueous phase was extracted with ethyl acetate (30mL×3). The organic layers were combined, washed with brine and driedover anhydrous sodium sulfate. The solvent was removed under reducedpressure to give the crude product. Further purification was conductedby Prep-HPLC to obtain the desired product (35 mg, 19%). ¹H-NMR (400MHz, DMSO-d⁶) δ(ppm) 15.85 (s, 1H), 10.75 (s, 2H), 8.88 (s, 1H), 8.34(d, J=3.7 Hz, 1H), 8.22 (d, J=2.5 Hz, 1H), 7.88 (d, J=2.1 Hz, 1H), 7.56(d, J=2.1 Hz, 1H). MS [MH]⁻ calcd for C₁₃H₇N₄O₅ 299.05, found 299.0.

Compound 525:

525 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of(E)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(pyrimidin-4-yl)acrylonitrile(2)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added tosolution of 2-(pyrimidin-4-yl)acetonitrile (0.44 g, 2.0 mmol) inanhydrous THF (10 mL) at −5° C. The resulting suspension was stirred at−5° C. for 15 min and the THF solution of 3,4-dimethoxy-5-nitrobenzoylchloride was added over 10 min and stirred for an additional 1 hour at−5° C. The reaction mixture was warmed to 0° C., quenched by theaddition of 1N.HCl solution (4 mL) and stirred for 10 min at roomtemperature, extracted by ethyl acetate (25 mL×2), the organic layerswas dried with Na₂SO₄ and concentrated in vacuo to give the titlecompound as an orange solid (550 mg, 85%). MS [MH]⁺ calcd for C₁₅H₁₂N₄O₅329.0 found 329.0.

Step 2: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(pyrimidin-4-yl)acrylonitrile(3)

A solution of(E)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(pyrimidin-4-yl)acrylonitrile(250 mg, 0.76 mmol) in DCM (5 mL) was added BBr₃ (0.5 mL, 5 mmol) at −5°C. under a nitrogen atmosphere. The resulting red suspension was stirredfor 1 h at −5° C. and allowed to warm to room temperature overnight. Thereaction was quenched by the addition of H₂O (2 mL) and stirred for 30min. The aqueous phase was extracted with ethyl acetate (30 mL×3). Theorganic layers were combined, washed with brine and dried over Na₂SO₄.The solvent was eliminated under reduced pressure to give the crudeproduct. Further purification by Prep-HPLC (0.5% TFA, MeOH/H₂O) gave thedesired product as a bright yellow solid (46 mg, 19%). ¹H NMR (400 MHz,DMSO) δ 15.35 (s, 1H), 10.67 (s, 2H), 8.95-7.16 (m, 5H). MS [MH]⁺ calcdfor C₁₅H₁₄N₂O₅ 337.0 found 301.0 (free).

Compound 503:

503 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

It Step 1: Synthesis of3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-2-(thiazol-2-yl)propanenitrile (2)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to thesolution of 2-(thiazol-2-yl) acetonitrile (0.24 g, 2.0 mmol) inanhydrous THF (5 mL) at −5° C. The resulting suspension was stirred at−5° C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoylchloride in THF was added over 10 min and stirred for an additional 1hour at −5° C. The reaction mixture was warmed to 0° C., quenched by theaddition of 1N.HCl solution (4 mL) and stirred for 10 min at roomtemperature, extracted by ethyl acetate (25 mL*2), the organic layerswas dried with Na₂SO₄ and concentrated in vacuo to give the titlecompound as an orange solid (240 mg, 39%). MS [MH]⁺ calcd forC₁₄H₁₁N₃O₅S 334.3, found 334.3.

Step 2: Synthesis of3-(3,4-dihydroxy-5-nitrophenyl)-3-oxo-2-(thiazol-2-yl)propanenitrile (3)

A solution of3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-2-(thiazol-2-yl)propanenitrile(224 mg, 0.67 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ inDCM (3 mL, 3 mmol) at −15° C. under nitrogen atmosphere. The resultingred suspension was stirred for 1 hour at −15° C. and allowed to warm toroom temperature overnight. The reaction was quenched by the addition ofH₂O (2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL×3). The organic layers were combined, washed withbrine and dried over Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLC(0.5% TFA, MeOH/H₂O) gave the desired product as a bright yellow solid(24 mg, 12%).¹H NMR (400 MHz, DMSO) δ 7.88 (d, J=2.0 Hz, 1H), 7.60 (d,J=4.0 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.31 (d, J=4.0 Hz, 1H). MS [MH]⁻calcd for C₁₂H₇N₃O₅S 304.0, found 304.0.

Compound 374:

374 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-N-(thiazol-2-yl)propanamide(2)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added tosolution of 2-cyano-N-(thiazol-2-yl)acetamide (0.35 g, 2.0 mmol) inanhydrous THF (10 mL) at −5° C. The resulting suspension was stirred at−5° C. for 15 min and the THF solution of 3,4-dimethoxy-5-nitrobenzoylchloride was added over 10 min and stirred for an additional 1 hour at−5° C. The reaction mixture was warmed to 0° C., quenched by theaddition of 1N.HCl solution (4 mL) and stirred for 10 min at roomtemperature, extracted by ethyl acetate (25 mL×2), the organic layerswas dried with Na₂SO₄ and concentrated in vacuo to give the titlecompound as an orange solid (510 mg, 67%). MS [MH]⁺ calcd forC₁₅H₁₂N₄O₆S 377.0, found 377.0.

Step 2: Synthesis of2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-3-oxo-N-(thiazol-2-yl)propanamide(3)

A solution of2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-N-(thiazol-2-yl)propanamide(400 mg, 1.1 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM(5 mL, 5 mmol) at −15° C. under a nitrogen atmosphere. The resulting redsuspension was stirred for 1 h at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL×3). The organic layers were combined, washed withbrine and dried over Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLC(0.5% TFA, MeOH/H₂O) gave the desired product as a bright yellow solid(100 mg, 28%). ¹H NMR (400 MHz, DMSO) δ 7.88 (d, J=2.0 Hz, 1H), 7.60 (d,J=4.0 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.31 (d, J=4.0 Hz, 1H). MS [MH]⁺calcd for C₁₃H₈N₄O₆S 349.0, found 349.0.

Compound 655:

655 was prepared in four synthetic steps from methyl5-chloropyrazine-2-carboxylate, according to the following procedure:

Step 1: Synthesis of methyl5-(2-(tert-butoxy)-1-cyano-2-oxoethyl)pyrazine-2-carboxylate (2)

A solution of tent-butyl 2-cyanoacetate (2.1 g, 15 mmol) and t-BuOK (1.6g, 15 mmol) in dry THF (50 mL) stirred at rt for 30 min, then methyl5-chloropyrazine-2-carboxylate (1.7 g, 10.0 mmol) was added, thereaction mixture was heated to reflux overnight, After the reaction wascompleted, cooled it to rt and quenched by H₂O (100 mL), the solid wasfiltered and dried in vacuo to afford the desired product as yellowsolid (1.7 g, 61%). MS [M+H]⁺ calcd for C₁₃H₁₅N₃O₄ 278.1, found 278.1.

Step 2: Synthesis of methyl 5-(cyanomethyl)pyrazine-2-carboxylate (3)

A solution of5-(2-(tert-butoxy)-1-cyano-2-oxoethyl)pyrazine-2-carboxylate (1.7 g,6.14 mmol) and p-TsOH (314 mg, 1.84 mmol) was heated to reflux for 3 h,then TLC showed no starting materials left. The reaction mixture wasquenched by H₂O (5 mL), extracted by EA, washed with sat. NaHCO₃, theorganic layer was dried with Na₂SO₄, filtered and dried in vacuo toafford the crude product, further purification by column chromatography(SiO₂, 100 g, 200-300 m, eluted by PE/EA=5/1) to afford the desiredproduct methyl 5-(cyanomethyl)pyrazine-2-carboxylate (800 mg, 74%) asyellow solid. MS [M+H]⁺ calcd for C₈H₇N₃O₂ 178.1 found 178.1.

Step 3: Synthesis of methyl (Z)-methyl5-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate(4)

A solution of methyl 5-(cyanomethyl)pyrazine-2-carboxylate (170 mg, 1.0mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1.0 mmol) and NH₄OAc(554 mg, 7.2 mmol) in MeOH (15 mL) was heated to reflux for 3 hours,then cooled it to room temperature. The solid was filtered and washed byH₂O (15 mL), The solid was re-dissolved in MeOH (5 mL). 5 mL of 1Naqueous HCl was added to till pH=3-4. The desired product was obtainedby filter and dried in vacuo as bright solid (210 mg, 61%). MS [M+H]⁺calcd for C₁₅H₁₀N₄O₆ 343.0, found 343.0.

Step 4: Synthesis of(Z)-5-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylicacid (5)

A solution of (Z)-methyl5-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate(150 mg, 0.44 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ inDCM (2 mL, 2 mmol) at −15° C. under a nitrogen atmosphere. Thesuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL×3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated in vacuo to givethe crude product. Further purification by Prep-HPLC (0.5% TFA,MeOH/H₂O) gave the desired product as bright yellow solid (55 mg, 38%).¹H NMR (300 MHz, DMSO) δ 9.27 (s, 1H), 9.22 (s,1H), 8.57 (s, 1H), 8.12(s, 1H), 7.98 (s, 1H), 2.52 (s, 19H), 0.02 (s, 1H). MS [MH]⁺ calcd forC₁₄H₈N₄O₆ 329.0, found 329.0.

Compound 656:

656 was prepared in two synthetic steps from 5-chloro-1,2,4-thiadiazole,according to the following procedure:

Step 1: Synthesis of 2-(1,2,4-thiadiazol-5-yl)acetonitrile (2)

A solution of dry MeCN (226 mg, 11 mmol) in dry THF (25 ml) was addedLiHMDS (5.5 mmol, 5.5 mL) at 0° C., then the mixture was stirred at 0°C. for 30 min, 5-chloro-1,2,4-thiadiazole (691 mg, 5.5 mmol) in dry THF(5 mL) was added to the mixture at 0° C., then stirred at rt overnight.The reaction mixture was quenched by H₂O (1 mL), and extracted by EA (30mL×3), dried with Na₂SO₄, filtered and dried in vacuo to afford thecrude product. Further purification by column chromatography (SiO₂, 100g, 200-300 m, eluted by PE/EA=5:1) gave the desired product (410 mg,60%) as yellow solid. MS [M+H]⁺ calcd for C₄H₃N₃S 126.0, found 126.0.

Step 2: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(1,2,4-thiadiazol-5-yl)acrylonitrile(3)

A solution of 2-(1,2,4-thiadiazol-5-yl)acetonitrile (150 mg, 1.2 mmol),3,4-dihydroxy-5-nitrobenzaldehyde (182 mg, 1 mmol) and NH₄OAc (462 mg, 6mmol) in MeOH (10 mL) was heated to reflux for 5 hours. LC-MS showed nostarting materials left. The solid was filtered and washed by MeO andH₂O, then dissolved in MeOH (5 mL). 5 ml of 1N.HCl was added tillpH=3-4. The solid was filtered and dried in vacuo to give the desiredproduct as bright yellow solid (200 mg, 69%). ¹H NMR (400 MHz, DMSO) δ10.96 (s, 1H), 8.97 (s, 1H), 8.45 (s, 1H), 8.14 (d, J=2.1 Hz, 1H), 7.95(d, J=2.2 Hz, 1H). MS [M+H]⁺ calcd for C₁₁H₆N₄O₄S 291.0, found 291.0.

Compound 660:

660 was prepared in two synthetic steps from(E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylic acid, according tothe following procedure:

Step 1: Synthesis of (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acryloylchloride (2)

A solution of (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylic acid(25 mg, 0.1 mmol) in DCM (25 ml) was added a drop of DMF and oxalyldichloride (25 mg,0.2 mmol). The reaction mixture was heated till thesolid dissolved, cooled to rt, concentrated in vacuo to dryness. It wasused in the next step without purification (27 mg, 100%).

Step 2: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(1,3-oxazinane-3-carbonyl)acrylonitrile(3)

A solution of 1,3-oxazinane (9 mg, 0.12 mmol), Et₃N (24mg, 0.24 mmol) inDCM (3 mL) was added acyl chloride dissolved in DCM (3 mL) dropwise at0° C., when the addition was completed, the reaction mixture was slowlywarmed to rt overnight. The reaction mixture was quenched by H₂O,separated the organic layer, and dried with Na₂SO₄, concentrated invacuo to afford the crude product, further purification by Prep-HPLC(0.5% TFA, MeOH/H₂O) afford the desired product as yellow solid (5 mg,16%). 1H NMR (400 MHz, DMSO) δ 10.87 (s, 2H), 7.95 (d, J=2.0 Hz, 1H),7.76 (d, J=2.1 Hz, 1H), 7.69 (s, 1H), 5.01 (s, 2H), 3.88-3.84 (m, 2H),3.74 (s, 2H), 1.72-1.66 (m, 2H). MS [M+H]+ calcd for C₁₄H₁₄N₃O₆ 320.1found. 319.9

Compound 661:

661 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of(Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl)acrylonitrile (2)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to thesolution of 3-oxo-3-(piperidin-1-yl)propanenitrile (0.30 g, 2.0 mmol) inanhydrous THF (5 mL) at −5° C. The resulting suspension was stirred at−5° C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoylchloride in THF was added over 10 min and stirred for an additional 1hour at −5° C. The reaction mixture was warmed to 0° C., quenched by theaddition of 1N.HCl solution (4 mL) and stirred for 10 min at roomtemperature, extracted by ethyl acetate (25 mL*2), the organic layerswas dried with Na₂SO₄ and concentrated in vacuo to give the titlecompound 3 as an orange solid (260 mg, 36%). MS [MH]⁺ calcd forC₁₇H₂₀N₃O₆ 362.1, found 362.1.

Step 2: Synthesis of(Z)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl)acrylonitrile(3)

A solution of(Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl)acrylonitrile(180 mg, 0.5 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM(3 mL, 3 mmol) at −15° C. under nitrogen atmosphere. The resulting redsuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL×3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLC(0.5% TFA, MeOH/H₂O) gave the desired product as yellow solid (63 mg,38%). 1H NMR (400 MHz, DMSO) δ 10.96 (s, 1H), 7.87 (d, J=2.0 Hz, 1H),7.49 (s, 1H), 3.65 (s, 4H), 1.60 (s, 6H). MS [M−H]-calcd for C₁₅H₁₆N₃O₆334.1, found 333.9

Compound 666:

666 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of 2-(cyanomethyl)thiazole-4-carboxylic acid (3)

Under a nitrogen atmosphere, 3-bromo-2-oxopropanoic acid in dry THF (5mL) was added to a suspension of 2-cyanoethanethioamide (1.2 g, 12 mmol)in THF (20 ml) at 0° C. The mixture was heated at 70° C. and stirred for3 hours. The aqueous phase was extracted with ethyl acetate (30 mL×3).The organic layers were combined, washed with brine and dried withNa₂SO₄. The solvent was eliminated under reduced pressure to give thecrude product. Further purification by column chromatography (SiO₂, 100g, 200-300 m, eluted by PE/EA=1/1) afforded the desired product as whitesolid (350 mg, 18%).

Step 2: Synthesis of(E)-2-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)thiazole-4-carboxylicacid (4)

A solution of 2-(cyanomethyl)thiazole-4-carboxylic acid (168 mg, 1.0mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1.0 mmol) and NH₄OAc(554 mg, 7.2 mmol) in MeOH (15 mL) was heated to reflux for 3 hours,then cooled to room temperature. The solid was filtered and washed byH₂O (15 mL), The solid was re-dissolved in MeOH (5 mL). 5 mL of 1Naqueous HCl was added to adjust pH 3-4. The desired product was obtainedby filter and dried in vacuo as bright solid (150 mg, 45%). ¹H NMR (400MHz, DMSO) δ 7.98 (s, 1H), 7.91 (s, 1H), 7.84 (s, 1H), 7.15-7.75 (m,4H). MS [MH]⁺ calcd for C₁₃H₇N₃O₆S 334.0, found 334.0.

Compound 668:

668 was prepared in three synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of 2-cyano-N-(1,2,4-thiadiazol-5-yl)acetamide (2)

Under a nitrogen atmosphere, NaH (200 mg, 5 mmol, 60%) was added to asuspension of 1,2,4-thiadiazol-5-amine (500 mg, 5 mmol) in THF (25 mL)at 0° C. The resulting suspension was stirred at 0° C. for 15 min andthe solution of 2-cyanoacetyl chloride (500 mg, 5 mmol) in THF was addedover 10 min and stirred for an additional 1 h at RT, then quenched bythe addition of 1N.HCl solution and stirred for 10 min at roomtemperature. Extracted it by ethyl acetate (25 mL×2), and the organiclayers was dried with Na₂SO₄ and concentrated in vacuo to give the crudeproduct. Washed it by PE/EA=1:1 (5 mL) to afford the purity product (260mg, 30%). MS [MH]⁺ calcd for C₅H₄N₄OS 169.0, found 169.0.

Step 2: Synthesis of2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-(1,2,4-thiadiazol-5-yl)acrylamide(3)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. When the reaction was complete, the organic solvent waseliminated by distillation under reduced pressure. Additional toluenewas added and eliminated again. The resulting yellowish solid3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5mL)

Under a nitrogen atmosphere, 60% NaH (80 mg, 2.0 mmol) was added tosolution of 2-cyano-N-(1,2,4-thiadiazol-5-yl)acetamide (250 mg, 1.5mmol) in anhydrous THF (10 mL) at −5° C. The resulting suspension wasstirred at −5° C. for 15 min and the THF solution of3,4-dimethoxy-5-nitrobenzoyl chloride was added over 10 min and stirredfor an additional 1 hour at −5° C. The reaction mixture was warmed to 0°C.; quenched by the addition of 1N.HCl solution (4 mL) and stirred for10 min at room temperature. Extracted by ethyl acetate (25 mL×2), theorganic layers was dried with Na₂SO₄ and concentrated in vacuo to givethe title compound as orange solid (210 mg, 37%). MS [M+H]⁺ calcd forC₁₄H₁₁N₅O₆S 378.0, found 378.0.

Step 3: Synthesis of2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-N-(1,2,4-thiadiazol-5-yl)acrylamide(4)

A solution of2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-(1,2,4-thiadiazol-5-yl)acrylamide(150 mg, 0.40 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ inDCM (2 mL, 2 mmol) at −15° C. under a nitrogen atmosphere. Thesuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL×3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLC(0.5% TFA, MeOH/H₂O) gave the desired product as bright yellow solid (50mg, 36%).'H NMR (400 MHz, DMSO) δ 13.80 (s, 1H), 8.33 (s, 1H), 7.72 (d,J=1.8 Hz, 1H), 7.47 (d, J=1.8 Hz, 1H).MS [MH]⁺ calcd for C₁₂H₇N₅O₆S350.0, found 350.0.

Compound 673:

673 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-(pyrimidin-4-ylmethyl)acrylamide(2)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to thesolution of 2-cyano-N-(pyrimidin-4-ylmethyl)acetamide (0.35 g, 2.0 mmol)in anhydrous THF (5 mL) at −5° C. The resulting suspension was stirredat −5° C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoylchloride in THF was added over 10 min and stirred for an additional 1hour at −5° C. The reaction mixture was warmed to 0° C., quenched by theaddition of 1N.HCl solution (4 mL) and stirred for 10 min at roomtemperature, extracted by ethyl acetate (25 mL*2), the organic layerswas dried with Na₂SO₄ and concentrated in vacuo to give the titlecompound 3 as an orange solid (169 mg, 22%). MS [M+H]⁺ calcd forC₁₇H₁₆N₅O₆ 386.1, found 386.0.

Step 2: Synthesis of(Z)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-N-(pyrimidin-4-ylmethyl)acrylamide(3)

A solution of(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-(pyrimidin-4-ylmethyl)acrylamide(115 mg, 0.3 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM(3 mL, 3 mmol) at −15° C. under nitrogen atmosphere. The resulting redsuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL×3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLC(0.5% TFA, MeOH/H₂O) gave the desired product as yellow solid (8 mg,7%). 1H NMR (400 MHz, DMSO) δ 14.18 (s, 1H), 10.74 (s, 2H), 9.12 (s,1H), 8.75 (d, J=5.2 Hz, 1H), 7.88 (s, 1H), 7.55 (d, J=2.1 Hz, 1H), 7.45(d, J=4.7 Hz, 1H), 4.52 (s, 2H). MS [M+H]⁺ calcd for C₁₅H₁₂N₅O₆ 356.1,found 356.0

Compound 675:

675 was prepared by four synthetic steps from methyl3-hydroxyisoxazole-5-carboxylate according to the following procedure:

Step 1: Synthesis of methyl 3-methoxyisoxazole-5-carboxylate (2)

A mixture of methyl 3-hydroxyisoxazole-5-carboxylate (1.0 g, 7.0 mmol)and K₂CO₃ (1.9 g, 14 mmol) in dry DMF (15 mL) was added Me₂SO₄ (1.0 g,8.4 mmol) at 0° C. The reaction solution was stirred at 0° C.continuously, and monitored by TLC until all the starting material wasconsumed completely; 50 mL of water was added. The residue was extractedby EA for two times (25 mL×2), and the organic layer was washed withbrine (25 mL×3), and dried over anhydrous Na₂SO₄. The organic layer wasconcentrated in vacuo to afford the desired product (850 mg, 77%)without further purification. MS [M+H]⁺ calcd for C₆H₇NO₄ 158.0, found158.0.

Step 2: Synthesis of3-(3-methoxyisoxazol-5-yl)-3-oxopropanenitrile (3)

A mixture of MeCN (553 mg, 13.5 mmol) and t-BuOK (1.5 g, 13.5 mmol) indry THF (25 mL) and toluene (15 mL) was added methyl3-methoxyisoxazole-5-carboxylate (850 mg, 5.4 mmol) at rt. The reactionsolution was stirred at 80° C. continuously for 24 h, and monitored byTLC until all the starting material was consumed completely. 50 mL ofwater was added. The aqueous phase was extracted by EA for two times (25mL×2), and the organic layer was combined and washed with brine (25mL×3), and dried over anhydrous Na₂SO₄. The organic layer wasconcentrated in vacuo to afford the crude product which was purified bysilica chromatograph (100 g, 200-300 m, eluted by PE/EA=3/1) to affordthe desired product as bright yellow solid (650 mg, 72%). MS [M+H]⁺calcd for C₇H₆N₂O₃167.0, found 167.0.

Step 3: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(3-methoxyisoxazole-5-carbonyl)acrylonitrile(5)

A mixture of 3-(3-methoxyisoxazol-5-yl)-3-oxopropanenitrile (200 mg, 1.2mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1.0 mmol) and NH₄OAc(554 mg, 7.2 mmol) in MeOH (15 mL) was refluxed for 3 hours, then cooledto room temperature. The reaction mixture was filtered and the solid wascollected and washed by H₂O (15 mL). The solid was re-dissolved in MeOH(5 mL) and the PH was adjusted to 3-5 by adding 5 mL of 1N aqueous HCl.The desired product was obtained by filter and dried in vacuo as brightyellow solid (250 mg, 76%). MS [MH]⁺ calcd for C₁₄H₉N₃O₇ 332.0, found332.0.

Step 4: Synthesis of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(3-hydroxyisoxazole-5-carbonyl)acrylonitrile(6)

A solution of(E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(3-methoxyisoxazole-5-carbonyl)acrylonitrile (150 mg, 0.45 mmol) in AcOH (5 mL) was added 0.5 mL 33%HBr (in AcOH) at rt. The reaction solution was stirred at 80° C.continuously for 3 h and monitored by LC-MS until all the startingmaterial was consumed completely. The reaction mixture was concentratedin vacuo to afford the crude product which was purified by by Prep-HPLC(0.5% TFA, MeCN/H₂O) to afford the desired product as yellow solid (22mg, 15%). MS [MH]⁺ calcd for C₁₃H₇N₃O₇ 318.0, found 318.0.¹H NMR (400MHz, DMSO) δ 11.96 (s, 2H), 8.32 (s, 1H), 8.18 (d, J=2.1 Hz, 1H), 7.88(d, J=2.2 Hz, 1H), 6.93 (s, 1H)

Compound 687:

687 was prepared in two synthetic steps from 2-cyanoacetate according,according to the following procedure:

Step 1: Synthesis of 3-(azetidin-1-yl)-3-oxopropanenitrile (2)

A mixture of ethyl 2-cyanoacetate (565 mg, 5.0 mmol) and azetidine (285mg, 5.0 mmol) was stirred at rt overnight. The reaction solution waspurified by HPLC to afford the desire product. MS [MH+H]⁺ calcd forC₆H₉N₂O₁ 125.1, found 125.1

Step 2: Synthesis of 3-(azetidin-1-yl)-3-oxopropanenitrile (3)

A mixture of 3-(azetidin-1-yl)-3-oxopropanenitrile (124 mg, 1.0 mmol),3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1.0 mmol) and CH₃COONH₄ (770mg, 10.0 mmol) in MeOH (15 mL) was stirred at 80° C. overnight and thencooled to rt. The reaction mixture was filtered to collect the solid.The solid was re-dissolved in MeOH and the PH was adjusted to 3-5 byadding 1N HCl. The desire product was collected by filtered and dried invavuo. (116 mg, 40%). MS [M+H]⁺ calcd for C₁₃H₁₂N₃O₅ 290.2, found 289.9.¹H NMR (400 MHz, DMSO) δ 10.93 (s, 2H), 8.01 (d, J=2.1 Hz, 1H), 7.97 (s,1H), 7.81 (d, J=2.1 Hz, 1H), 4.48 (t, J=7.2 Hz, 2H), 4.03 (t, J=7.3 Hz,2H), 2.32-2.22 (m, 2H).

Compound 688:

688 was prepared in two synthetic steps from 2-cyanoacetate according tothe following procedure:

Step 1: Synthesis of 2-cyano-N-(2-hydroxyethyl)acetamide (2)

A mixture of ethyl 2-cyanoacetate (113 mg, 1.0 mmol) and 2-aminoethanol(305 mg, 5.0 mmol) was stirred at rt overnight. The reaction solutionwas purified by HPLC to afford the desire product. MS [M+H]⁺ calcd forC₅H₉N₂O₂ 129.1, found 129.1

Step 2: Synthesis of(E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-(2-hydroxyethyl)acrylamide(3)

A mixture of 2-cyano-N-(2-hydroxyethyl)acetamide (65 mg, 0.5 mmol),3,4-dihydroxy-5-nitrobenzaldehyde (92 mg, 0.5 mmol) in MeOH (15 mL) andCH₃COONH₄ (385 mg, 5.0 mmol) was stirred at 80° C. overnight and thencooled to rt. The reaction mixture was filtered to collect the solid.The solid was re-dissolved in MeOH and the PH was adjusted to 3-5 byadding 1N HCl. The desire product was collected by filtered and dried invavuo. (80 mg, 54%). MS [M+H]⁺ calcd for C₁₃H₁₂N₃O₆ 294.2, found 293.8.¹H NMR (400 MHz, DMSO) δ 10.92 (s, 2H), 8.30 (t, J=5.5 Hz, 1H), 8.06 (s,1H), 7.95 (d, J=2.1 Hz, 1H), 7.78 (d, J=2.2 Hz, 1H), 3.49 (t, J=6.1 Hz,2H), 3.28 (q, J=5.9 Hz, 2H), 3.17 (s, 1H).

Compound 691:

691 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-ethylacrylamide(3)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to thesolution of 2-cyano-N-ethylacetamide (0.23 g, 2.0 mmol) in anhydrous THF(5 mL) at −5° C. The resulting suspension was stirred at −5° C. for 15min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF wasadded over 10 min and stirred for an additional 1 h at −5° C. Thereaction mixture was warmed to 0° C., quenched by the addition of 1N.HClsolution (4 mL) and stirred for 10 min at room temperature, extracted byethyl acetate (25 mL*2), the organic layers was dried with Na₂SO₄ andconcentrated in vacuo to give the title compound 3 as an yellow solid(288 mg, 39%).

Step 2: Synthesis of(Z)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-ethyl-3-hydroxyacrylamide(4)

A solution of(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-ethylacrylamide(167 mg, 0.5 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM(3 mL, 3 mmol) at −15° C. under nitrogen atmosphere. The resulting redsuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL*3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLCgave the desired product as yellow solid (48 mg, 33%). 1H NMR (400 MHz,DMSO) δ 14.18 (s, 1H), 10.74 (s, 2H), 9.12 (s, 1H), 8.75 (d, J=5.2 Hz,1H), 7.88 (s, 1H), 7.55 (d, J=2.1 Hz, 1H), 7.45 (d, J=4.7 Hz, 1H), 4.52(s, 2H). MS [M+H]⁺ calcd for C₁₂H₁₂N₃O₆ 294.2, found 293.8

Compound 692:

692 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of(Z)-2-cyano-N-cyclopropyl-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylamide(3)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to thesolution of 2-cyano-N-cyclopropylacetamide (250 mg, 2.0 mmol) inanhydrous THF (5 mL) at −5° C. The resulting suspension was stirred at−5° C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoylchloride in THF was added over 10 min and stirred for an additional 1 hat −5° C. The reaction mixture was warmed to 0° C., quenched by theaddition of 1N.HCl solution (4 mL) and stirred for 10 min at roomtemperature, extracted by ethyl acetate (25 mL*2), the organic layerswas dried with Na₂SO₄ and concentrated in vacuo to give the compound 3as an yellow solid (242 mg, 36%).

Step 2: Synthesis of(Z)-2-cyano-N-cyclopropyl-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxyacrylamide(4)

A solution of(Z)-2-cyano-N-cyclopropyl-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylamide(166 mg, 0.5 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM(3 mL, 3 mmol) at −15° C. under nitrogen atmosphere. The resulting redsuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL*3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLCgave the desired product as yellow solid (25 mg, 26%). 1H NMR (400 MHz,DMSO) δ 10.81 (s, 1H), 8.82 (s, 1H), 7.88 (d, J=1.9 Hz, 1H), 7.54 (d,J=2.1 Hz, 1H), 2.83-2.77 (m, 1H), 0.74-0.64 (m, 4H). MS [M−H]⁻ calcd forC₁₃H₁₀N₃O₆ 304.2, found 303.9.

Compound 697:

697 was prepared in two synthetic steps from methyl5-(cyanomethyl)pyrazine-2-carboxylate, according to the followingprocedure:

Step 1: Synthesis of (E)-methyl5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylate(3)

Under a nitrogen atmosphere, 60% NaH (0.072 g, 1.8 mmol) was added tothe solution of methyl 5-(cyanomethyl)pyrazine-2-carboxylate (0.15 g,0.9 mmol) in anhydrous THF (5 mL) at −5° C. The resulting suspension wasstirred at −5° C. for 15 min and the solution of3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min andstirred for an additional 1 hour at −5° C. The reaction mixture waswarmed to 0° C., quenched by the addition of 1N.HCl solution (4 mL) andstirred for 10 min at room temperature, extracted by ethyl acetate (25mL*2), the organic layers was dried with Na₂SO₄ and concentrated invacuo to give the title compound (E)-methyl5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylateas an orange solid(125 mg, 30%). MS [M+H]⁺ calcd for C₁₇H₁₄N₄O₇ 387.0,found 387.0.

Step 2: Synthesis of(E)-5-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylic acid (4)

A solution of (E)-methyl5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylate (125 mg, 0.3 mmol) in DCM (5 mL) was added 1.0 Msolution of BBr₃ in DCM (3 mL, 3 mmol) at −15° C. under nitrogenatmosphere. The resulting red suspension was stirred for 1 hour at −15°C. and allowed to warm to room temperature overnight. The reaction wasquenched by the addition of H₂O (2 mL) and stirred for 30 min. Theaqueous phase was extracted with ethyl acetate (30 mL*3). The organiclayers were combined, washed with brine and dried with Na₂SO₄. Thesolvent was eliminated under reduced pressure to give the crude product.Further purification by Prep-HPLC (0.5% TFA, MeOH/H₂O) gave the desiredproduct as yellow solid (15 mg, 15%). 1H NMR (400 MHz, DMSO) δ 10.57 (s,1H), 9.17 (s, 1H),8.68 (s, 1H), 7.81 (s, 1H), 7.51 (s, 1H). MS [M+H]⁺calcd for C₁₄H₈N₄O₇ 345.0, found 345.0

Compound 701:

701 was prepared in two synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of(Z)-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylonitrile(3)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to thesolution of 2-cyano-N-cyclopropylacetamide (250 mg, 2.0 mmol) inanhydrous THF (5 mL) at −5° C. The resulting suspension was stirred at−5° C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoylchloride in THF was added over 10 min and stirred for an additional 1 hat −5° C. The reaction mixture was warmed to 0° C., quenched by theaddition of 1N.HCl solution (4 mL) and stirred for 10 min at roomtemperature, extracted by ethyl acetate (25 mL*2), the organic layerswas dried with Na₂SO₄ and concentrated in vacuo to give the compound 3(146 mg, 22%).

Step 2: Synthesis of(Z)-2-(azetidine-1-carbonyl)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxyacrylonitrile(4)

A solution of(Z)-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylonitrile(146 mg, 0.4 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM(2 mL, 2 mmol) at −15° C. under nitrogen atmosphere. The resulting redsuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL*3). The organic layers were combined, washed withbrine and dried with Na2SO4. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLCgave the desired product (18 mg, 15%). 11H NMR (400 MHz, DMSO) δ 10.93(s, 2H), 7.93 (d, J=2.1 Hz, 1H), 7.55 (d, J=2.1 Hz, 1H), 4.36 (s, 4H),2.35-2.27 (m, 2H). MS [M+H]+ calcd for C13H12N306 306.2, found 305.9

Compound 711:

711 was prepared in two synthetic steps from3-oxo-3-(thiazol-4-yl)propanenitrile, according to the followingprocedure:

Step 1: Synthesis of(Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile(2)

Under a nitrogen atmosphere, 60% NaH (0.072 g, 1.8 mmol) was added tothe solution of 3-oxo-3-(thiazol-4-yl)propanenitrile (0.41 g, 2.7 mmol)in anhydrous THF (15 mL) at −5° C. The resulting suspension was stirredat −5° C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoylchloride (661 mg, 2.7 mmol) in THF was added over 5 min and stirred foran additional 1 hour at −5° C. The reaction mixture was warmed to 0° C.,quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10min at room temperature, extracted by ethyl acetate (25 mL*2), theorganic layers was dried with Na₂SO₄ and concentrated in vacuo to givethe title compound (E)-methyl5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylateas an orange solid (600 mg, 62%). MS [M+H]+ calcd for C₁₅H₁₁N₃O₆S 362.0,found 362.0.

Step 2: Synthesis of(Z)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile(4)

A solution of(Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile(70 mg, 0.2 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM(1 mL, 1 mmol) at −15° C. under nitrogen atmosphere. The resulting redsuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of 0.5N.NH₄OH (2 mL) and stirred for 30 min. The aqueous phase was extractedwith ethyl acetate (30 mL*3). The organic layers were combined, washedwith brine and dried with Na₂SO₄. The solvent was eliminated underreduced pressure to give the crude product. Further purification byPrep-HPLC (0.5% TFA, MeOH/H₂O) gave the desired product as yellow solid(11 mg, 17%). 1H NMR (400 MHz, DMSO) δ 10.66 (s, 1H), 8.61 (s,1H), 7.97(s, 1H), 7.65 (s, 1H). MS [M+H]⁺ calcd for C₁₃H₇N₃O₆S 334.0, found334.0.

Compound 709:

709 was prepared in two synthetic steps from(E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylic acid, according tothe following procedure:

Step 1: Synthesis of (E)-methyl2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)acetate (2)

A solution of (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylic acid(1) (250 mg, 1 mmol) and methyl 2-aminoacetate (89 mg, 1 mmol) in THF(15 mL) was added HBTU (600 mg, 1.5 mmol) and DIPEA (388 mg, 3 mmol) atrt. The resulting suspension was stirred for 5 hours at 60° C. andallowed to cool to room temperature overnight. The reaction was addedH₂O and extracted with ethyl acetate (30 mL*3). The organic layers werecombined to afford the crude product which was used in the next stepwithout further purification (120 mg, 37.4%).

Step 2: Synthesis of(E)-2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)acetic acid (3)

A solution of (E)-methyl2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)acetate (2) (120mg, 0.37 mmol) in THF/H₂O (5 mL/5 mL) was added LiOH (24 mg, 0.55 mmol)at 0° C. The resulting suspension was stirred for 1 hour at 0° C. andwas quenched by adding aqueous HCl. The aqueous phase was extracted withethyl acetate (30 mL*3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLCgave the desired product (7 mg, 6.2%). 1H NMR (400 MHz, DMSO) δ 8.65 (t,J=5.8 Hz, 1H), 8.10 (s, 1H), 7.98 (d, J=2.1 Hz, 1H), 7.79 (d, J=2.1 Hz,1H), 3.88 (d, J=5.8 Hz, 2H). MS [M+H]⁺ calcd for C₁₂H₁₀N₃O₇ 308.2, found308.0.

Compound 693:

693 was prepared in three synthetic steps from ethyl 2-cyanoacetate,according to the following procedure:

Step 1: Synthesis of (S)-methyl 2-(2-cyanoacetamido)-3-hydroxypropanoate(2)

A mixture of ethyl 2-cyanoacetate (1130 mg, 10.0 mmol) and (S)-methyl2-amino-3-hydroxypropanoate (1190 mg, 10.0 mmol) was stirred at r.t.overnight. The reaction solution was added 10 mL MeOH and filtered tocollect the solid as crude product, which was used in next step withoutfurther purification. MS [M+H]^(±) calcd for C₇H₁₁N₂O₄ 187.2, found187.2.

Step 2: Synthesis of (S,E)-methyl2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoate(3)

A solution of (S)-methyl 2-(2-cyanoacetamido)-3-hydroxypropanoate (930mg, 5 mmol) (2) and 3,4-dihydroxy-5-nitrobenzaldehyde (915 mg, 5 mmol)in MeOH (25 mL) was added CH₃COONH₄ (3850 mg, 50 mmol) at rt. Thereaction solution was stirred at 60° C. continuously for 5h andmonitored by TLC until all the starting material was consumedcompletely. Then the reaction mixture was cooled to rt and the solventwas eliminated under reduced pressure, then the Sat. aq. NaCl (100 mL)was added. The aqueous solution was extracted by EA for three times (50mL*3). The organic layer was concentrated in vacuo to afford crudeproduct which was purified by silica chromatograph chromatography toafford the desired product (705 mg, 40%). MS [M+H]⁺ calcd for C₁₄H₁₄N₃O₈352.3, found 352.3.

Step 3: Synthesis of(S,E)-2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoicacid (4)

A solution of (S,E)-methyl2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoate(3) (705 mg, 2 mmol) in THF/H₂O (10 mL/10 mL) was added LiOH (126 mg, 3mmol) at 0° C. The resulting suspension was stirred for 1 hour at 0° C.and was quenched by adding aqueous HCl. The aqueous phase was extractedwith ethyl acetate (30 mL*3). The organic layers were combined, washedwith brine and dried with Na₂SO₄. The solvent was eliminated underreduced pressure to give the crude product. Further purification byPrep-HPLC gave the desired product (220 mg, 32.6%). 1H NMR (400 MHz,DMSO) δ 12.89 (s, 1H), 10.94 (s, 2H), 8.22 (d, J=7.6 Hz, 1H), 8.15 (s,1H), 7.97 (d, J=2.1 Hz, 1H), 7.82 (d, J=2.1 Hz, 1H), 5.05 (s, 1H), 4.41(m, 1H), 3.79 (m, 2H). MS [M+H]⁺ calcd for C₁₃H₁₂N₃O₈ 338.2, found 337.8

Compound 702:

702 was prepared in three synthetic steps from ethyl 2-cyanoacetate,according to the following procedure:

Step 1: Synthesis of (S)-methyl1-(2-cyanoacetyl)pyrrolidine-2-carboxylate (2)

A mixture of ethyl 2-cyanoacetate (113 mg, 1.0 mmol) and (S)-methylpyrrolidine-2-carboxylate (129 mg, 1.0 mmol) was stirred at r.t.overnight. The reaction solution was added 10 mL MeOH and filtered tocollect the solid as crude product, which was used in next step withoutfurther purification. MS [M+H]⁺ calcd for C₉H₁₃N₂O₃197.2, found 197.2.

Step 2: Synthesis of (S,E)-methyl2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoate(3)

A solution of (S)-methyl 1-(2-cyanoacetyl)pyrrolidine-2-carboxylate (2)(196 mg, 1 mg) and 3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1 mmol) inMeOH (5 mL) was added CH₃COONH₄ (770 mg, 10 mmol) at rt. The reactionsolution was stirred at 60° C. overnight and monitored by TLC until allthe starting material was consumed completely. Then the reaction mixturewas cooled to r.t. and the solvent was eliminated under reducedpressure, then the Sat. aq. NaCl (100 mL) was added. The aqueoussolution was extracted by EA for three times (50 mL*3). The organiclayer was concentrated in vacuo to afford crude product which waspurified by silica chromatograph chromatography to afford the desiredproduct (43 mg, 12%). MS [M+H]⁺ calcd for C₁₆H₁₆N₃O₇362.3, found 362.3.

Step 3: Synthesis of(S,E)-1-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acryloyl)pyrrolidine-2-carboxylicacid (4)

A solution of (S,E)-methyl2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoate(3) (43 mg, 0.12 mmol) in THF/H₂O (2 mL/1 mL) was added LiOH (8 mg, 0.18mmol) at 0° C. The resulting suspension was stirred for 1 hour at 0° C.and was quenched by adding aqueous HCl. The aqueous phase was extractedwith ethyl acetate (30 mL*3). The organic layers were combined, washedwith brine and dried with Na₂SO₄. The solvent was eliminated underreduced pressure to give the crude product. Further purification byPrep-HPLC gave the desired product (6 mg, 15%). 1H NMR (400 MHz, DMSO) δ7.25 (t, J=7.4 Hz, 1H), 7.21-7.09 (m, 2H), 2.30 (s, 2H), 2.07 (s, 4H).MS [M+H]⁺ calcd for C₁₅H₁₄N₃O₇ 348.3, found 347.8

Compound 347N:

347N was prepared in single synthetic step from2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide (1,compound 347), according to the following procedure:

Step 1: Synthesis of2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-hydroxyacrylamide(2)

A solution of2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide(350 mg, 1 mmol) in DCM (5 mL) was added HMDS (0.5 mL) at rt, thenstirred for 48 h. After the reaction was completed, the reaction mixturewas concentrated in vacuo to afford the crude product, furtherpurification by Prep-HPLC afforded the desired product as yellow solid(60 mg, 19%). MS [M+H]⁺ calcd for C₁₄H₁₆N₄O₅ 321.1, found 321.1.

Compound 661N:

661N was prepared in one synthetic step from(Z)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl)acrylonitrile,according to the following procedure:

Step 1: Synthesis of3-amino-3-(3,4-dihydroxy-5-nitrophenyl)-2-(piperidine-1-carbonyl)acrylonitrile(2)

A solution of3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl)acrylonitrile(20 mg, 0.06 mmol) in DCM (5 mL) was added HMDS (1 mL) and stirred at rtfor 72 h. And then the reaction mixture was concentrated in vacuo toafford the crude product, which was further purified by HPLC to affordthe desired product as a yellow solid (1 mg, 5%). MS [MH]+ calcd forC₁₅H₁₆N₄O₅ 333.3, found 333.1. 1H NMR (400 MHz, DMSO) δ 8.21 (s, 1H),7.66 (s,1H), 6.74 (s, 1H), 1.25-1.65 (m, 6H), 1.23 (s, 1H).

Compound 691N:

691N was prepared in four synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-ethylacrylamide(3)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to thesolution of 2-cyano-N-ethylacetamide (0.23 g, 2.0 mmol) in anhydrous THF(5 mL) at −5° C. The resulting suspension was stirred at −5° C. for 15min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF wasadded over 10 min and stirred for an additional 1 hour at −5° C. Thereaction mixture was warmed to 0° C., quenched by the addition of 1N.HClsolution (4 mL) and stirred for 10 min at room temperature, extracted byethyl acetate (25 mL*2), the organic layers was dried with Na₂SO₄ andconcentrated in vacuo to give the title compound 3 as an yellow solid(265 mg, 36%).

Step 2: Synthesis of(Z)-3-chloro-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N-ethylacrylamide(4)

A solution of(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-ethylacrylamide(176 mg, 0.5 mmol) in DCM (5 mL) was added PCl₅ (104 mg, 0.5 mmol) at 0°C. The resulting suspension was stirred at overnight until all the startmaterials were consumed detected by LC-MS. Then the reaction mixture wasallowed to cool to room temperature and used in the next step withoutfurther purification.

Step 3: Synthesis of(Z)-3-amino-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N-ethylacrylamide(5)

A solution of(Z)-3-chloro-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N-ethylacrylamide(150 mg, 0.4 mmol) in DCM was added a saturated solution of NH₃ in ACNat 0° C., and then the reaction solution was stirred at 0° C.continuously until all the starting materials were consumed completely.The reaction was quenched by the addition of H₂O and stirred for 30 min.The aqueous phase was extracted with ethyl acetate. The organic layerswere combined, washed with brine and dried with Na₂SO₄. The solvent waseliminated under reduced pressure to give the crude product.

Step 4: Synthesis of(Z)-3-amino-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-ethylacrylamide(6)

A solution of(Z)-3-amino-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N-ethylacrylamide(67 mg, 0.2 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM(1 mL, 1 mmol) at −15° C. under nitrogen atmosphere. The resulting redsuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL*3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLCgave the desired product as yellow solid (5 mg, 9%). 1H NMR (400 MHz,DMSO) δ 10.53 (s, 2H), 9.44 (s, 1H), 8.02 (s, 2H), 7.63 (d, J=2.0 Hz,1H), 7.36 (d, J=2.0 Hz, 1H), 3.32-3.25 (m, 2H), 1.14 (t, J=7.1 Hz, 3H).MS [M+H]⁺ calcd for C₁₂H₁₃N₄O₅ 293.2, found 292.8.

Compound 692N:

692N was prepared in four synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of(Z)-2-cyano-N-(cyclopropylmethyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylamide(3)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to thesolution of 2-cyano-N-cyclopropylacetamide (250 mg, 2.0 mmol) inanhydrous THF (5 mL) at −5° C. The resulting suspension was stirred at−5° C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoylchloride in THF was added over 10 min and stirred for an additional 1hour at −5° C. The reaction mixture was warmed to 0° C., quenched by theaddition of 1N.HCl solution (4 mL) and stirred for 10 min at roomtemperature, extracted by ethyl acetate (25 mL*2), the organic layerswas dried with Na₂SO₄ and concentrated in vacuo to give the titlecompound 3 as an yellow solid(165 mg, 23%).

Step 2: Synthesis of(Z)-3-chloro-2-cyano-N-(cyclopropylmethyl)-3-(3,4-dimethoxy-5-nitrophenyl)acrylamide(4)

A solution of(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-propylacrylamide(165 mg, 0.5 mmol) in DCM (5 mL) was added PCl₅ (104 mg, 0.5 mmol) at 0°C. The resulting suspension was stirred at overnight until all the startmaterials were consumed detected by LC-MS. Then the reaction mixture wasallowed to cool to room temperature and used in the next step withoutfurther purification.

Step 3: Synthesis of(Z)-3-amino-2-cyano-N-cyclopropyl-3-(3,4-dimethoxy-5-nitrophenyl)acrylamide(5)

A solution of(Z)-3-chloro-2-cyano-N-(cyclopropylmethyl)-3-(3,4-dimethoxy-5-nitrophenyl)acrylamide(164 mg, 0.5 mmol) in DCM was added a saturated solution of NH₃ in ACNat 0° C., and then the reaction solution was stirred at 0° C.continuously until all the starting materials were consumed completely.The reaction was quenched by the addition of H₂O and stirred for 30 min.The aqueous phase was extracted with ethyl acetate. The organic layerswere combined, washed with brine and dried with Na₂SO₄. The solvent waseliminated under reduced pressure to give the crude product which wasused in the next step without further purification.

Step 4: Synthesis of(Z)-3-amino-2-cyano-N-cyclopropyl-3-(3,4-dihydroxy-5-nitrophenyl)acrylamide(6)

A solution of(Z)-3-amino-2-cyano-N-cyclopropyl-3-(3,4-dimethoxy-5-nitrophenyl)acrylamide(66 mg, 0.2 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM(1 mL, 1 mmol) at −15° C. under nitrogen atmosphere. The resulting redsuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL*3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLCgave the desired product. (7 mg, 12%). 1H NMR (400 MHz, DMSO) δ 10.52(s, 2H), 9.53 (s, 1H), 7.63 (d, J=2.0 Hz, 1H), 7.35 (d, J=2.0 Hz, 1H),2.61-2.55 (m, 1H), 0.86-0.81 (m, 2H), 0.66-0.61 (m, 2H). MS [M+H]⁺ calcdfor C₁₃H₁₃N₄O₅ 305.3, found 304.9

Compound 697N:

697N was prepared in two synthetic steps from (E)-methyl5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylate,according to the following procedure:

Step 1: Synthesis of (E)-methyl 5-(2-chloro-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate (2)

A solution of (E)-methyl5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylate (150 mg, 0.39 mmol) in 1,2-dichloroethane (25 mL)was added PCl₅ (83 mg, 0.40 mmol) at 0° C. The resulting suspension wasstirred at overnight until all the start materials were consumeddetected by LC-MS. Then the reaction mixture was allowed to cool to roomtemperature and used in the next step without further purification.

Step 2: Synthesis of (E)-methyl 5-(2-amino-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate (3)

A solution of (E)-methyl 5-(2-chloro-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl) pyrazine-2-carboxylate (180mg, 0.45 mmol) in 1,2-dichloroethane (25 mL) was added a saturatedsolution of NH₃ in ACN at 0° C., and then the reaction solution wasstirred at 0° C. continuously until all the starting materials wereconsumed completely. The reaction was quenched by the addition of H₂Oand stirred for 30 min. The aqueous phase was extracted with ethylacetate. The organic layers were combined, washed with brine and driedwith Na₂SO₄. The solvent was eliminated under reduced pressure to givethe crude product which was used in the next step without furtherpurification.

Step 3: Synthesis of (E)-methyl5-(2-chloro-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate(4)

A solution of (E)-methyl 5-(2-amino-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl) pyrazine-2-carboxylate (98mg, 0.25 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM (3mL, 3 mmol) at −15° C. under nitrogen atmosphere. The resulting redsuspension was stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL*3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLC(0.5% TFA, MeOH/H₂O) gave the desired product as yellow solid (11 mg,13%). 1H NMR (400 MHz, DMSO) δ 10.96-11.2 (m, 1H), 10.42 (s, 1H), 8.94(s, 1H), 8.66 (s, 1H), 7.76 (s, 1H), 6.79 (s, 1H). MS [M+H]⁺ calcd forC₁₄H₉N₅O₆ 344.0, found 344.0

Compound 701N:

701N was prepared in four synthetic steps from3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

Step 1: Synthesis of(Z)-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylonitrile(3)

Under a nitrogen atmosphere, SOCl₂ (0.38 mL, 5.28 mmol) and anhydrousDMF (0.01 mL, 0.22 mmol) were added to a suspension of3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL)at room temperature. The mixture was heated at 60° C. and stirred for 15hours. The organic solvent was eliminated by distillation under reducedpressure. More toluene was added and eliminated again. The resultingyellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved inanhydrous THF (5 mL).

Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to thesolution of 3-(azetidin-1-yl)-3-oxopropanenitrile (250 mg, 2.0 mmol) inanhydrous THF (5 mL) at −5° C. The resulting suspension was stirred at−5° C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoylchloride in THF was added over 10 min and stirred for an additional 1hour at −5° C. The reaction mixture was warmed to 0° C., quenched by theaddition of 1N.HCl solution (4 mL) and stirred for 10 min at roomtemperature, extracted by ethyl acetate (25 mL*2), the organic layerswas dried with Na₂SO₄ and concentrated in vacuo to give the compound 3.

Step 2: Synthesis of(Z)-2-(azetidine-1-carbonyl)-3-chloro-3-(3,4-dimethoxy-5-nitrophenyl)acrylonitrile(4)

A solution of(Z)-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylonitrile(175 mg, 0.5 mmol) in DCM (5 mL) was added PCl₅ (104 mg, 0.5 mmol) at 0°C. The resulting suspension was stirred at overnight until all the startmaterials were consumed detected by LC-MS. Then the reaction mixture wasallowed to cool to room temperature and used in the next step withoutfurther purification.

Step 3: Synthesis of(Z)-3-amino-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)acrylonitrile(5)

A solution of(Z)-2-(azetidine-1-carbonyl)-3-chloro-3-(3,4-dimethoxy-5-nitrophenyl)acrylonitrile(164 mg, 0.5 mmol) in DCM was added a saturated solution of NH₃ in ACNat 0° C., and then the reaction solution was stirred at 0° C.continuously until all the starting materials were consumed completely.The reaction was quenched by the addition of H₂O and stirred for 30 min.The aqueous phase was extracted with ethyl acetate. The organic layerswere combined, washed with brine and dried with Na₂SO₄. The solvent waseliminated under reduced pressure to give the crude product which wasused in the next step without further purification.

Step 4: Synthesis of(Z)-3-amino-2-(azetidine-1-carbonyl)-3-(3,4-dihydroxy-5-nitrophenyl)acrylonitrile(6)

A solution of (Z)-3-amino-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)acrylonitrile (66 mg, 0.2mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ in DCM (1 mL, 1mmol) at −15° C. under nitrogen atmosphere. The resulting red suspensionwas stirred for 1 hour at −15° C. and allowed to warm to roomtemperature overnight. The reaction was quenched by the addition of H₂O(2 mL) and stirred for 30 min. The aqueous phase was extracted withethyl acetate (30 mL*3). The organic layers were combined, washed withbrine and dried with Na₂SO₄. The solvent was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLCgave the desired product (7 mg, 12%). 1H NMR (400 MHz, DMSO) δ 10.52 (s,2H), 9.87 (s, 1H), 7.60 (d, J=2.1 Hz, 1H), 7.35 (d, J=2.1 Hz, 1H), 4.26(s, 4H), 2.29 (m, 2H). MS [M+H]⁺ calcd for C₁₃H₁₃N₄O₅ 305.3, found 305.0

Compound 711N:

711N was prepared in three synthetic steps from(Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile,according to the following procedure:

Step 1: Synthesis of(Z)-3-chloro-3-(3,4-dimethoxy-5-nitrophenyl)-2-(thiazole-4-carbonyl)acrylonitrile(2).

A solution of(Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile(150 mg, 0.42 mmol) in 1,2-dichloroethane (25 mL) was added PCl₅ (86 mg,0.42 mmol) at 0° C. The resulting suspension was stirred at overnightuntil all the start materials were consumed detected by LC-MS. Then thereaction mixture was allowed to cool to room temperature and used in thenext step without further purification.

Step 2: Synthesis of(Z)-3-amino-3-(3,4-dimethoxy-5-nitrophenyl)-2-(thiazole-4-carbonyl)acrylonitrile(3)

A solution of(Z)-3-chloro-3-(3,4-dimethoxy-5-nitrophenyl)-2-(thiazole-4-carbonyl)acrylonitrile(185 mg, 0.42 mmol) in 1,2-dichloroethane (25 mL) was added a saturatedsolution of NH₃ in ACN at 0° C., and then the reaction solution wasstirred at 0° C. continuously until all the starting materials wereconsumed completely. The reaction was quenched by the addition of H₂Oand stirred for 30 min. The aqueous phase was extracted with ethylacetate. The organic layers were combined, washed with brine and driedwith Na₂SO₄. The solvent was eliminated under reduced pressure to givethe crude product which was used in the next step without furtherpurification. MS [M+H]⁺ calcd for C₁₅H₁₂N₄O₅S 361.0, found 361.0

Step 3: Synthesis of ammonium(Z)-5-(1-amino-2-cyano-3-oxo-3-(thiazol-4-yl)prop-1-enyl)-2-hydroxy-3-nitrophenolate(4)

A solution of(Z)-3-amino-3-(3,4-dimethoxy-5-nitrophenyl)-2-(thiazole-4-carbonyl)acrylonitrile(100 mg, 0.28 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ inDCM (1 mL, 1 mmol) at −15° C. under nitrogen atmosphere. The resultingred suspension was stirred for 1 hour at −15° C. and allowed to warm toroom temperature overnight. The reaction was quenched by the addition of0.5 N.NH₄OH (2 mL) and stirred for 5 min. The aqueous phase was washedwith ethyl acetate. The hydrous layer was eliminated under reducedpressure to give the crude product. Further purification by Prep-HPLC(0.05% FA, MeOH/H₂O) gave the desired product as yellow solid (11 mg,15%). 1H NMR (400 MHz, DMSO) δ 8.85 (s, 1H), 7.66 (s, 1H), 7.38 (s, 1H),6.80-6.99 (m, 1H), 6.66-6.74 (m, 1H). MS [M+H]⁺ calcd for C₁₃H₁₁N₅O₅S333.0, found 333.0

Compound 347M:

347M was prepared in three synthetic steps from2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-hydroxyacrylamideaccording to the following procedure:

Step 1: Synthesis of2-cyano-3-(diethylamino)-1-(3,4-dimethoxy-5-nitrophenyl)-3-oxoprop-1-enylmethanesulfonate (2)

A solution of2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-hydroxyacrylamide(350 mg, 1 mmol) and Et₃N (202 mg, 2 mmol) in DCM (15 mL) was added MsCl(250 mg, 2 mmol) at 0° C. The reaction solution was stirred at rt for 3h, then it was concentrated in vacuo to afford the crude product (360mg), which was used in the next step without further purification.

Step 2: Synthesis of2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-(methylamino)acrylamide(3)

A solution of2-cyano-3-(diethylamino)-1-(3,4-dimethoxy-5-nitrophenyl)-3-oxoprop-1-enylmethanesulfonate (250 mg, 0.7 mmol) and K₂CO₃ (193 mg, 1.4 mmol) in MeCN(10 mL) was added MeNH₂ (1.4 mmol, 0.7 mL, 2N in THF) at rt. Thereaction solution was refluxed for 2 h, and then 50 mL of water wasadded. The aqueous phase was extracted by EA for two times (50 mL×2),and then the organic layer was concentrated in vacuo to afford the crudeproduct (210 mg), which was used in the next step without furtherpurification. MS [M+H]⁺ calcd for C₁₇H₂₂N₄O₅363.1., found 363.1.

Step 3: Synthesis of2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-(methylamino)acrylamide(4)

A solution of2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-(methylamino)acrylamide(210 mg, 0.58 mmol) in DCM (5 mL) was added 1.0 M solution of BBr₃ inDCM (2 mL, 2 mmol) at −15° C. under nitrogen atmosphere. The reactionsolution was stirred at −15° C. for 1 h then at room temperatureovernight. The reaction was quenched by the addition of H₂O (2 mL) andstirred for 30 min. The aqueous phase was extracted by ethyl acetate forthree times (30 mL×3). The organic layer was washed with brine and driedover Na₂SO₄, then it was concentrated in vacuo to afford the crudeproduct, which was purified by Prep-HPLC (0.5% TFA, MeOH/H₂O) to gainthe desired product as bright yellow solid (25 mg, 13%). MS ¹H NMR (400MHz, DMSO) δ 10.69 (s, 2H), 7.43 (s, 1H), 7.20(s, 1H), 3.40-3.43 (m,4H), 2.69 (d, J=4.9 Hz, 3H), 1.14 (t, J=6.8 Hz, 13H), 1.14 (t, J=6.8 Hz,3H). [M+H]⁺ calcd for C₁₂H₇N₅O₆S 350.0, found 350.0.

Enzymatic Inhibition.

We measured compound inhibition activity in a demethylation reactioncatalyzed by FTO (US2014/0148383A1). The reaction system was incubatedat 37° C. for 2 h and stopped by heating at 95° C. for 5 min. ssDNA wasdigested by nuclease P1 and alkaline phosphatase. The concentrations ofN⁶-mA and A were analyzed by HPLC-MS/MS. When concentration of substrateand enzyme are 0.5 μM and 0.1 μM, respectively, the measured IC₅₀ valueof entacapone against FTO is ˜3 μM. The compounds of Tables 1-3 wereconsistently active, with IC₅₀'s less than 10 μM, and most less than 1μM.

Exemplary IC50 Data of submicromolar representative compounds CompoundNo. Enzymatic inhibition (IC50) μM 687 <1 317 >1 371 ~1 660 <1 382 >1702 ~1 698 >1 675 >1 394 >1 664 ~1 684 >1 688 >1 713 <1 709 <1 712 ~1693 ~1 331 ~1 333 >1 318 ~1 365 ~1 366 >1 390 ~1 656 <1 666 <1 315 ~1319 <1 389 ~1 502 <1 505 <1 395 <1 396 <1 522 ~1 655 <1 518 >1 520 <1347 <1 351 <1 523 <1 524 ~1 525 <1 503 ~1 359 >1 374 <1 668 <1 661 ~1673 <1 674 >1 691 <1 692 <1 697 <1 701 <1 711 ~1 715 <1 722 <1 347N <1661N <1 691N ~1 692N >1 697N <1 701N >1 711N >1 347M ~1

In vivo Anti-Obesity Efficacy.

Male wistar rats (6 weeks) were fed with high-fat diet (45% fat,OpenSource Diets D12451), and compound (100 mg/kg) was administered to12 randomly selected rats by gavage. After 8 weeks, the mean body weightof drug treatment group was less than that of control group. However,the body-weight-normalized food intakes of the two groups showed nodifference. The LDL-c (Low Density Lipoprotein—cholesterol) in serum ofdrug treatment group was reduced compared to that of control group, andthe adipose and hepatic tissues of rat in drug-treated groups showedreduced size of adipose cells and reduced level of liver steatosis.

Atherosclerosis Model: Ldlr-Deficient Mice.

We measured compound anti-atherosclerosis efficacy using Ldlr^(−/−) micefed western style diet (20% fat, 0.15% cholesterol), compound (100mg/day) was orally administered by blending with diet. After 8 weeks,the mean lesion area in aortic sinuses of drug treatment group was lessthan that of the control group.

In Vivo Anti-Obesity Efficacy in Obese Mice.

Male C57BL/6 mice were fed with high-fat diet (45% fat, OpenSource DietsD12451) for 8 weeks. Then obese mice with body weight 20% larger thanthat of mice fed with normal diet (20 mice) were selected forexperiments. Compound (100 mg/kg) was orally administered to 10 randomlyselected obese mice by blending with diet. After 13 weeks, the mean bodyweight gain of drug treatment group was about less than that of thecontrol group.

In Vivo Anti-Diabetic Efficacy in Genetically Diabetic db/db Mice.

Compound (100 mg/kg) was orally administered to 11 randomly selectedmale db/db mice by blending with normal diet, while the other 9 maledb/db mice fed with normal diet as control group. After 20 weeks, themean fasting plasma glucose of drug treatment group was significantlylower than that of control group (*p-value<0.05).

From our results herein and further data we determine that preferredanti-weight gain, anti-obesity and anti-obesity related disease, such asanti-diabetes, effective human dosages of the compounds should be0.1-10, 0.5-10, 0.5-5, 0.5-2.5, 0.5-1, 1-10, 1-5, 1-2.5, 2-10, or 2-5g/day.

COMT Inhibition Assay.

We measured compound COMT inhibitory activity by reaction kinetic model.The test article was diluted with assay buffer to desired concentration.The COMT enzyme was also diluted with assay buffer. Then 5 μL dilutedtest article, 5 μL diluted COMT and 5 μL Esculetin were added into plateand incubated for 5 mM at 37° C., sealed with TopSeal-A 384, ClearAdhesive (PE). Then 5 μL AdoMet was added into the plate. The reactionsystem contains 1U COMT enzyme, test article, 4 μM Esculetin, 0.6 mMAdoMet, 50 mM K₃PO₄, and 10 mM MgCl₂. Read plate by using kinetics model(Excitation at 360 nm & emission at 460 nm). The inhibition wascalculated from the slope.

Rat Liver Microsome Stability Assay.

Compound stability was tested in rat liver microsome. Microsome workingsolution contains rat liver microsome 1 mg/ml, test compound 2 μM, NADPH1 mM in 0.05 M Phosphate buffer (pH=7.4). This reaction system wasincubated at 37° C. At each time point of 0, 5, 15, 30 and 60 mM, analiquot of 15 μL was transferred into another tube with 200 μL quenchingsolution (Internal standard (Terfenadine) 5 ng/ml in Methanol). Vortex,Centrifuge at 3,500 rpm for 12 min, Transfer 100 μL of supernatant to 96well plate for LC-MS/MS analysis and dilute sample with MeOH: H2O (1:1)if necessary.

Result of Selectivity and Rat Liver Microsome Selectivity Rat LiverMicrosome Compound FTO (μM) COMT(μM) Index* T½ (min) CLint (μL/min/mgprotein) Entacapone 3 0.06  1.00 39.2 17.7 347 0.5 0.551 55.10 69310.100 315 1 70% at 200 nM; 2.24 310 19% at 50 nM 351 0.75 80% at 200 nM;77.0 9.00 33% at 50 nM 396 0.25 0.108 21.60 33.2 20.9 523 0.5 0.10510.50 239 2.90 395 0.5 74% at 200 nM; 55.0 12.6 36% at 50 nM 525 0.5 74%at 200 nM; 63.6 10.9 39% at 50 nM 390 1 36% at 200 nM; 2.61 266 12% at50 nM 503 1.5 24% at 200 nM; 1386 0.50 9% at 50 nM 331 1 54% at 200 nM;93.7 7.40 25% at 50 nM 374 0.5 0.065 6.50 1155 0.600 371 1 71% at 200nM; 16.1 43.0 33% at 50 nM 661 1 26% at 200 nM; 315 2.20 12% at 50 nM655 0.3 0.024 4.00 433 1.60 697 0.75 33% at 200 nM; 630 1.10 −3 at 50 nM687 1 0.033 1.65 2.44 284 701 0.75 3.56  237.33 NA NA Entacapone 3 0.06 1.00 39.2 17.7 347N 0.5 0.214 21.40 141 4.90 371 1 71% at 200 nM; 16.143.0 33% at 50 nM 661N 0.75 64% at 200 nM; 67.3 10.3 35% at 50 nMEntacapone 3 0.06  1.00 39.2 17.7 347M 1 0.162 8.10 56.8 12.2*Selectivity Index is the normalized COMT/FTO value based onEntacapone's value and was calculated as the following equation:(COMT_(compound)/FTO_(compound))/(COMT_(entacapone)/FTO_(entacapone))

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein, including citations therein, are herebyincorporated by reference in their entirety for all purposes.

What is claimed is:
 1. An FTO inhibitor selected from a compound formulaI, a stereoisomer thereof, a hydride thereof, and apharmaceutically-acceptable salt thereof, or a pharmaceuticalcomposition formulated in unit dosage and suitable for administration toa person in need thereof, the inhibitor of structure:

wherein: (a) R1 and R2 are independently H or Me; R3 is OH or NHR,wherein R is H or an optionally substituted, optionally hetero-,optionally cyclic C1-C18 hydrocarbyl; and R4 is optionally substituted,optionally hetero-, optionally cyclic C1-C18 hydrocarbyl; (b) R1 and R2are independently H or Me; R3 is H, OH or NHR, wherein R is H or C1-C4alkyl; R4 is CONHR5; and R5 is optionally substituted, optionallyhetero-, optionally cyclic C1-C18 hydrocarbyl; (c) R1 and R2 areindependently H or Me; R3 is H, OH or NHR, wherein R is H or C1-C4alkyl; R4 is COR5; and R5 is optionally substituted, heterocyclic C3-C18hydrocarbyl comprising an n-membered ring wherein n=3-18 including 1 ton-1 heteroatoms independently selected from N, O, S and P; or (d) R1 andR2 are independently H or Me; R3 is H, OH or NHR, wherein R is H orC1-C4 alkyl; and R4 is optionally substituted, heterocyclic C3-C18hydrocarbyl comprising an n-membered ring wherein n=3-18 including 1 ton-1 heteroatoms independently selected from N, O, S and P; whereinexcluded from the inhibitor, unless present in the composition, arecompounds 309, 365, 371 and
 361. 2. The inhibitor or composition ofclaim 1 wherein the heterocyclic C3-C18 hydrocarbyl comprises: a 3membered ring that is an optionally substituted: aziridine, oxirane,oxaziridine; a 4 membered ring that is an optionally substituted:azetidine, oxetane, oxazetidine; a 5 membered ring that is an optionallysubstituted: pyrrole, 1,2-diazole (pyrazole), 1,3 diazole (imidazole),thiazole, isothiazole, oxazole, isoxazole, furan, dioxole, thiophene; a6 membered ring that is an optionally substituted: pyridine, diazine,triazine, oxazine, thiazine, dioxine, oxathiine, dithiine; a 9 memberedring that is an optionally substituted: indole, benzothiazole,benzooxazole, benzofuran, benzodioxole, benzothiophene, benzodithiole;or a 10 membered ring that is an optionally substituted: quinoline,quinoxaline, quinazoline, chromene, benzodioxine, thiochromene,benzodithiine.
 3. The inhibitor or composition of claim 1 wherein theoptionally substituted, optionally hetero-, optionally cyclic C1-C18hydrocarbyl in each instance is an optionally substituted C1-C9 alkyl,C2-C9 alkenyl, C2-C9 alkynyl, or C5-C14 aryl hydrocarbon, comprising 1-5heteroatoms that are N, S, O or P, including 1-5 nitrogen atoms, or aheteroatom substituted with the hydrocarbon.
 4. The inhibitor orcomposition of claim 1 wherein: one or both R1 and R2 is H; R3 is OH;and/or R is H or C1-C4 alkyl.
 5. A compound of formula I

or a stereoisomer thereof, a hydride thereof, or apharmaceutically-acceptable salt thereof, wherein (a) R₁ and R₂ are eachindependently H or C₁₋₄alkyl; R₃ is OH or NHR, wherein R is hydrogen,—C₁₋₄alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; R₄ ishydrogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, —SO₂R_(a), —COR_(a), —CO₂R_(a),—CONR_(a)R_(b), NR_(a)R_(b), —NR_(a)COR_(b), —NR_(a)CO₂R_(b), or—NR_(a)SO₂R_(b); wherein said —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl are each independentlyoptionally substituted with at least one substituent R_(c); whereinR_(a) and R_(b) are each independently hydrogen, C₁₋₄alkyl,heteroarylC₁₋₄alkyl-, heterocyclylC₁₋₄alkyl-, aryl, heteroaryl, orC₃₋₆cycloalkyl; or R_(a) and R_(b), together with the atom(s) to whichthey are attached form a 3- or 4- or 5- or 6-membered ring optionallycomprising an additional heteroatom selected from the group of O, NH, Sand P; and R_(c) is hydrogen, halogen, —C₁₋₆alkyl, —C₂₋₆alkenyl,—C₂₋₆alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —CN, —NO₂,oxo, —OH, C₁₋₆alkyloxy, —SO₂H, C₁₋₆alkylSO₂—, —COH, C₁₋₆alkylCO—, CO₂H,C₁₋₆alkylCO₂—, CONH₂ or —NH₂; or (b) R₃ is OH; R₄ is heteroaryl,—COR_(a), or —CONR_(a)R_(b); wherein said heteroaryl is optionallysubstituted with at least one substituent R_(c); wherein R_(a) and R_(b)are each independently hydrogen, C₁₋₄alkyl, heteroarylC₁₋₄alkyl-,heterocyclylC₁₋₄alkyl-, aryl, heteroaryl, or C₃₋₆cycloalkyl; or R_(a)and R_(b), together with the nitrogen atom to which they are attachedform a 3- or 4- or 5- or 6-membered ring optionally comprising anadditional heteroatom selected from the group of O, NH, S and P; andR_(c) is hydrogen, halogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, —CN, —NO₂, oxo, —OH,C₁₋₆alkyloxy, —SO₂H, C₁₋₆alkylSO₂—, —COH, C₁₋₆alkylCO—, CO₂H,C₁₋₆alkylCO₂—, CONH₂ or —NH₂; provided that said compound is not(Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-hydroxyacrylamide.6. The compound of claim 5, wherein R₃ is OH; R₄ is heteroaryl,—COR_(a), or —CONR_(a)R_(b); wherein said heteroaryl is optionallysubstituted with at least one substituent R_(c); wherein R_(a) and R_(b)are each independently hydrogen, C₁₋₄alkyl, heteroarylC₁₋₄alkyl-,heterocyclylC₁₋₄alkyl-, aryl, heteroaryl, or C₃₋₆cycloalkyl; or R_(a)and R_(b), together with the nitrogen atom to which they are attachedform a 3- or 4- or 5- or 6-membered ring optionally comprising anadditional heteroatom selected from the group of O, NH, S and P; andR_(c) is hydrogen, halogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, —CN, —NO₂, oxo, —OH,C₁₋₆alkyloxy, —SO₂H, C₁₋₆alkylSO₂—, —COH, C₁₋₆alkylCO—, CO₂H,C₁₋₆alkylCO₂—, CONH₂ or —NH₂;
 7. The compound of claim 5, wherein: R₁and R₂ are each H; one of R₁ and R₂ is H, the other is C₁₋₄alkyl,preferably methyl; or R₁ and R₂ are each C₁₋₄alkyl, preferably methyl.8. The compound of claim 5, wherein R₄ is —COR_(a), wherein R_(a) isheteroaryl.
 9. The compound of claim 5, wherein R₄ is —CONR_(a)R_(b),wherein R_(a) and R_(b) are each independently hydrogen, C₁₋₄alkyl,heteroarylC₁₋₄alkyl-, heterocyclylC₁₋₄alkyl-, heteroaryl, orC₃₋₆cycloalkyl; preferably, R_(a) and R_(b) are both C₁₋₄alkyl; morepreferably, R_(a) and R_(b) are both ethyl.
 10. The compound of claim 5,wherein R₄ is —CONR_(a)R_(b), wherein one of R_(a) and R_(b) ishydrogen, the other is C₁₋₄alkyl, heteroarylC₁₋₄alkyl-,heterocyclylC₁₋₄alkyl-, heteroaryl or C₃₋₆cycloalkyl; preferably, one ofR_(a) and R_(b) is hydrogen, the other is C₁₋₄alkyl,pyrimidinylC₁₋₄alkyl- (e.g., pyrimidin-4-ylmethyl), 5- to 10-memberedheteroaryl (e.g., pyridinyl, pyrizinyl, pyrimidinyl, thiazolyl,benzo[d]thiazolyl, thiadiazolyl), or C₃₋₆cycloalkyl (e.g., cyclopropyl,cyclobutyl, cyclopentyl or cyclohexyl).
 11. The compound of claim 5,wherein R₄ is -CONR_(a)R_(b), wherein R_(a) and R_(b), together with thenitrogen atom to which they are attached form a 3- or 4- or 5- or6-membered ring optionally comprising one additional oxygen atom;preferably, R_(a) and R_(b), together with the nitrogen atom to whichthey are attached form a 4- or 6-membered ring optionally comprising oneadditional oxygen atom; more preferably, R_(a) and R_(b), together withthe nitrogen atom to which they are attached form a 4-membered ring, ora 6-membered ring or a 6-membered ring comprising one additional oxygenatom; most preferably, R_(a) and R_(b), together with the nitrogen atomto which they are attached form a piperidinyl, azetidinyl or1,3-oxazinanyl.
 12. The compound of claim 5, wherein R₄ is heteroaryloptionally substituted with at least one substituent R_(c), whereinR_(c) is as defined above; preferably, R₄ is a 5- or 6-memberedmonocyclic heteroaryl comprising one or two or three or four heteroatomsselected from NH, O, S or P; or a 9 or 10-membered bicylic heteroarylcomprising one or two or three or four heteroatoms selected from NH, O,S or P; more preferably, R₄ is pyridinyl, pyrizinyl optionallysubstituted by carboxyl, pyrimidinyl, thiazolyl, benzo[d]thiazolyl, or1,2,4-thiadiazolyl; most preferably, R₄ is pyridin-2-yl, pyrizin-2-yloptionally substituted by carboxyl, pyrimidin-4-yl, thiazol-2-yl,benzo[d]thiazol-2-yl, or 1,2,4-thiadiazol-5-yl.
 13. The compound ofclaim 5, wherein R₃ is OH; and R₄ is —CONR_(a)R_(b), wherein Ra and Rbare each C₁₋₄alkyl.
 14. The inhibitor or composition of claim 1,subsection (a), wherein R3 is OH, of formula:


15. The inhibitor or composition of claim 1, subsection (a), wherein R3is NHR, of formula:


16. The inhibitor or composition of claim 1, subsection (b), wherein R4is CONHR5, of formula:


17. The inhibitor or composition of claim 1, subsection (c), wherein R4is COR5, of formula:


18. The inhibitor or composition of claim 1, subsection (d), wherein R4is heterocyclic, of formula:


19. An inhibitor or claim 1 formulated in a pharmaceutical compositionin unit dosage and suitable for administration to a person.
 20. Use ofan inhibitor or composition of claim 1 in a person in need thereof, toinhibit FTO, inhibit weight gain, promote weight loss, reduce serum LDL,cholesterol, LDL-c, or triglycerides, or treat obesity or an obesityrelated disease or Alzheimer's disease.